Retinal pigmented epithelium derived neurotrophic factor and methods of use

ABSTRACT

The present invention relates to a purified retinal pigmented epithelium derived neurotrophic factor composition and a method for purifying such a retinal pigmented epithelium neurotrophic factor. The present invention also relates to a recombinant DNA molecule comprising a gene encoding a retinal pigmented epithelium derived neurotrophic factor having the DNA sequence or the amino acid sequence in SEQ ID NO:1 and to an organism transformed with the recombinant DNA molecule. 
     In addition, the present invention relates to a method of treating tumors, ocular diseases, nerve injuries, and conditions resulting from the activity of serine proteases, which comprises administering PEDF.

RELATED APPLICATIONS

This is a continuation of application Ser. No. 08/377,710, filed on Jan.25, 1995, abandoned, which is a continuation application of applicationSer. No. 08/279,979, filed on Jul. 25, 1994, abandoned, which is acontinuation-in-part application of application Ser. No. 07/894,215,filed on Jun. 4, 1992, abandoned, and a continuation-in-part applicationof application Ser. No. 07/952,796, filed Sep. 24, 1992, abandoned,which are incorporated herein by reference.

This invention was made with government support under grant EY04741,awarded by The National Institutes of Health. The United Statesgovernment has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates to the purification and use of a retinalpigmented epithelium derived neurotrophic factor (PEDF). This inventionalso relates to a truncated version of PEDF that is referred to asPEDF-BH. In addition to PEDF and PEDF-BH and functionally equivalentproteins, this invention relates to nucleic acids that encode PEDF,PEDF-BH and functionally equivalent proteins, to vectors comprising suchnucleic acids, to host cells into which such vectors have beenintroduced, and to the use of these host cells to produce such proteins.

BACKGROUND OF THE INVENTION

Many types of neurons depend upon the availability of special regulatorymolecules, known as neurotrophic factors, for their survival andwell-being. The best characterized of the neurotrophic factors is nervegrowth factor (NGF). NGF regulates the survival and specialized functionof sympathetic and dorsal root ganglion neurons in the peripheralnervous system and of some cholinergic neurons in the central nervoussystem. Trophic factors, which act on other neurons, have also beenidentified, and two such factors, ciliary neurotrophic factor (CNTF) andbrain-derived neurotrophic factor (BDNF) have been purified. Moreover,it has recently been shown that some growth factors, such as fibroblastgrowth factor (FGF) and epidermal growth factor (EGF), which initiallywere identified based on their mitogenic effects upon cells, alsofunction as survival-promoting agents for some neurons. Post-synaptictarget cells and satellite cells, such as glial cells, appear to bemajor sources of neurotrophic factors.

It has been proposed that the survival of retinal photoreceptor cellsmay also be regulated by specific neurotrophic factors. Evidencesupporting this concept includes the observation that photoreceptorsundergo developmental neuronal death in some species, a phenomenon whichis generally considered to reflect the limited availability ofneurotrophic factors. Photoreceptor development, as well as maintenanceof normal function, has also been shown to require interactions with theretinal pigment epithelium (RPE), suggesting that RPE-derived moleculesor factors could be necessary for photoreceptor function and survival.

The RPE develops in advance of and lies adjacent to the neural retina. Aclosed compartment between the two cell layers contains theinterphotoreceptor matrix, and many soluble secretory products of RPEand neural retina cells are contained in the interphotoreceptor matrix.Nutrients, metabolites or trophic factors exchanged between the RPE andneural retina, must pass through the interphotoreceptor matrix. RPEcells, for example, are thought to synthesize and secrete aphotoreceptor survival-promoting factor (PSPA).

Cultured RPE cells synthesize a number of well known trophic factors,including platelet derived growth factor (PDGF), FGF, transforminggrowth factor-α (TGF-α), and transforming growth factor-β (TGF-β). It ispossible that these or other unknown factors derived from RPE couldinfluence the development of the neural retina.

The neural-derived RPE forms a monolayer of cells interposed between theneural retina and circulating blood within the choroid. In thisstrategic location, the RPE forms a part of the blood-retina barrier,performs functions essential to retinal integrity and functions, andplays important roles in vascular, inflammatory, degenerative, anddystrophic diseases of the retina and choroid. The functions of the RPEin relation to the visual process are several-fold and includelight-dark adaption, phagocytosis of shed photoreceptor outer segmentmembrane and nutrition. On the other hand, the close interdependence ofthe RPE and the neural retina during normal development has been knownfor a long time, but functionally is not well understood, although it isknown that the RPE is important for retinal regeneration. It has beenconsistently observed that loss of contact of the neural retina with theRPE of many vertebrates (retinal detachment) results in degeneration ofthe retina. As a side effect of the retinal detachment, strong cellproliferation, originating from the RPE which underlies the areas ofdetachment, has often been observed.

Thus, identification of hypothetical survival-promoting factors forphotoreceptor cells would potentially be of great importance for thetreatment of pathological conditions which result in blindness due tophotoreceptor degeneration of unknown etiology. While these types ofselective photoreceptor degenerations could be due to a variety ofdifferent mechanisms, analogies with neuronal degenerations in otherregions of the nervous system suggest the possible involvement of aneurotrophic activity in the retina.

SUMMARY OF THE INVENTION

The present invention relates to a purified retinal pigmented epitheliumderived neurotrophic factor composition and a method for purifying sucha retinal pigmented epithelium neurotrophic factor. The purificationprocedure comprises providing an impure protein fraction containingretinal pigmented epithelium derived neurotrophic factor and applyingthe impure protein fraction containing retinal pigmented epitheliumderived neurotrophic factor to a cation-exchange chromatography medium.The cation-exchange chromatography medium is then washed to elute anyunbound proteins and the retinal pigmented epithelium derivedneurotrophic factor is eluted from the cation-exchange chromatographymedium and collected.

The present invention also relates to a recombinant DNA moleculecomprising a gene encoding a retinal pigmented epithelium derivedneurotrophic factor having the DNA sequence or the amino acid sequencein SEQ ID NO:1 and to an organism transformed with a recombinant DNAmolecule comprising a retinal pigmented epithelium derived neurotrophicfactor gene having a DNA sequence identified in SEQ ID NO:1.

The present invention also relates to a method of treating tumors,ocular diseases and conditions resulting from the activity of serineproteases which comprises administering PEDF.

DETAILED DESCRIPTION

Cells of the retinal pigmented epithelium are closely associated withdifferentiating retinoblasts in vivo and contribute to an environmentessential to their development and normal function, both in vivo and invitro. Retinoblastoma cells exhibit a multi-potential differentiativenature paralleling that of their precursor, the primitive retinoblast,as evidenced by the fact that agents, such as laminin, sodium butyrateand dibutyryl cAMP, can induce the expression of neuronal, glial, andpigmented epithelial characteristics in vitro.

A human retinoblastoma cultured cell line, Y79 cells, when exposed toRPE-conditioned medium (RPE-CM), exhibits a high degree of neuronal-likedifferentiation. The differentiation is observed in both themorphological and biochemical characteristics of the cells. The exposedcells extend arborizing neuritic processes from their cell bodies andexpress elevated levels of neuron-specific enolase (NSE) andneurofilament proteins. In addition, RPE-CM extends the life of theattached, differentiated cells for more than 30 days as compared to 10to 15 days for non-treated cells.

RPE-secreted proteins have been fractionated from RPE-CM, and a proteindoublet, with an apparent molecular weight of about 50,000 to about55,000, that is unique to RPE-CM has been identified. This pigmentepithelium derived neurotrophic factor (PEDF), which is a majorsecretory product of human fetal RPE cells, has been isolated and shownto have neurotrophic effects on Y79 retinoblastoma cells. Additionally,a smaller 36,000 molecular weight, form of PEDF has also beenidentified.

PEDF has uses in the treatment of retinal diseases including, but notlimited to, retinoblastoma and other ocular tumors, retinitispigmentosa, various forms of retinal detachment, macular degeneration,diabetic retinopathy, and other inherited and age-related pathologies ofretinal cells.

In the case of retinal tumors, PEDF induces the tumor cells to displaybiochemical and phenotypic characteristics of mature neuronal cells.Such changes are identified by a cessation or reduction in the rate ofcell division, which leads to tumor regression or a slowing in the rateof tumor growth. In the case of non-tumorous retinal diseases, theneurotrophic properties of PEDF enhance survival and well-being ofphotoreceptor or other retinal cells, prolonging their functional lifespan and delaying the rate of the onset of impaired vision and ultimateblindness. In the case of retinal detachment, PEDF prolongs the lifespan of photoreceptor cells sufficiently to allow standard reattachmentprocedures to be effective in re-establishing the retina-RPE interface,thereby restoring normal vision.

1. Preparation of an Impure Protein Fraction Containing RetinalPigmented Epithelium Derived Neurotrophic Factor

Retinal pigmented epithelium derived neurotrophic factor (PEDF) may beisolated from any tissue or cell producing PEDF.

Naturally-occurring cells that produce PEDF are retinal pigmentedepithelium cells. Such cells may be grown in culture to secrete PEDFinto the medium in which they are growing. The medium may be harvestedperiodically, and the PEDF isolated from the media. Suitable cellcultures may be established from retinal pigmented epithelium cellsderived from humans, monkeys, and other primates or other animals, suchas chickens, mice, rats, cows and pigs. PDEF may also be isolated fromthe vitreous humor of human, bovine, monkey and other primates. Thevitreous contains an abundance of PEDF and is very easy to remove fromthe eye cup. This is probably the easiest source from which to isolatePEDF.

PEDF may also be isolated by extraction of the interphotoreceptor matrix(IPM) or from the retina of humans, monkeys, and other primates or otheranimals, such as chickens, mice, rats, cows and pigs.

Alternatively PEDF may be derived from sources in which it is notnaturally-occurring, such as organisms transfected with a recombinantDNA molecule constructed to result in the expression of PEDF or a PEDFequivalent protein in the host cells chosen for the expression of thegene. It is well known in the art that proteins can be modified bydeletion, addition or substitution of amino acids without changing thefunction of the protein. Such proteins which retain the specificity ofPEDF are considered to be equivalent.

A. Culturing of Human Retinal Pigmented Epithelium (RPE) Cells

Cultures of RPE cells are established by harvesting post-mortem eyes byaseptically opening the eyes and removing the vitreous body and retina.The exposed RPE is washed with a buffered solution such as modifiedEarle's balanced salt solution (MEBS: 115.5 mM NaCl, 3.5 mM KCl, 1 mMNaH₂PO₄, 0.5 mM CaCl₂, 0.27 mM MgCl₂, 0.37 MgSO₄, 15 mM HEPES, 14 mMNaHCO₃, 12 mM glucose, pH 7.2). RPE cells are scraped from Bruch'smembrane or are dislodged by a stream of fluid such as MEBS or cellculture medium, applied using a Pasteur or similar pipette. This stepmay be preceded by exposure to proteolytic or other enzyme(s), such asDispase (Boehringer-Mannheim, Indianapolis, Ind., Catalog #295 825).Alternatively, RPE cells may be isolated by detaching sections of sclerafrom an intact eye to expose choroidal tissue and treating thischoroidal surface with proteolytic or other enzymes such as Dispaseprior to removal of the vitreous and retina and dislodging RPE cells bythe spraying of cell culture medium as described by Pfeffer et al., J.Cell. Physiol. 117 333-341 (1983). Tissue fragments are transferred totissue culture dishes in a medium such as “low Ca⁺⁺” or “high Ca⁺⁺”complete RPE-47 medium, as described by Pfeffer et al., J. Cell.Physiol. 117 333-341 (1983), or Eagles's minimal essential medium (MEM,supplied by GIBCO of Grand Island, N.Y.) supplemented with about 0.5% toabout 20% v/v fetal calf serum. At concentrations below about 0.5% v/vfetal calf serum, the serum concentrations are too low to effectivelysupport the growth of the cells, At concentrations above about 20% v/vserum, no additional benefit, with respect to cell growth, is conferredon the cells.

The medium may also be supplemented with antibiotics and/or fungicidesto prevent the growth of bacteria and/or fungi in the cultures.Antibiotics and fungicides suitable for use in the present invention areabout 1,000 units/ml penicillin, about 100 μg/ml streptomycin, about0.25 μg amphotericin, and about 50 μg/ml gentamicin, or other suitablesuch agents known in the art. These antibiotics may be used individuallyor in combination, as desired.

The cells are incubated at about 37° C. in an atmosphere of about 5% v/vC₂. Retinal pigmented epithelium (RPE) cells attach to the surface ofthe dishes, proliferate, and eventually form confluent monolayers.

When the cells have grown to confluence, about 3-7 days from the initialculturing of the cells, they are harvested, for example, by trypsinizingthe cell monolayer, or by other methods known to those skilled in theart, and resuspending the cells in a medium such as MEM, supplied byGIBCO, supplemented with about 5% v/v to about 20% v/v fetal calf serum.A portion of the cells are reseeded into sterile tissue culture flasks,after which time the cells are again grown in an atmosphere of about 5%v/v CO₂ at about 37° C.

Alternatively, RPE cells may be isolated by removing the cornea, the 2mm scleral ring, and the vitreous and neural retina from the eyes. Theeyes are washed with calcium and magnesium-free Hanks Balanced SaltSolution (HBSS: 1.3 mM CaCl₂, 5 mM KCl, 0.3 mM KH₂PO₄, 0.5 mM MgCl₂, 0.4mM MgSO₄, 138 mM NaCl, 4 mM NaHCO₃, 0.3 mM Na₂HPO₄, 5.6 mM D-glucose and0.03 mM Phenol Red, supplied by GIBCO/BRL of Gaithersburg, Md.). The eyecup is filled with a solution comprising about 0.1% w/v trypsin, about0.1% w/v hyaluronidase in calcium and magnesium-free Hanks balanced saltsolution, and the eye is incubated at about 37° C. for about 15 to about30 minutes.

The loose RPE cells are collected by gentle aspiration, and theprocedure is repeated until the RPE cells are released. The trypsin isinactivated by adding about 5% v/v to about 20% v/v fetal calf serum tothe cell sample. The cells are collected by centrifugation at about1,200 rpm for about 7 minutes.

The RPE cells are then plated onto sterile tissue culture plates at adensity of about 1×10⁵ cells for a 35 mm plate. (Proportionally more orless cells are plated if larger or smaller plates or containers areused.) The cells are grown in DulBecco's Modified Eagles Medium (DMEM),supplied by GIBCO, or other suitable medium. The medium may besupplemented with an equal volume of HAM's F12 medium (supplied byGIBCO), —about 1 mM sodium pyruvate, about 0.625 mM Hepes, about 6 mML-glutamine, about 1% w/v non-essential amino acids, about 5 μg/mlinsulin, about 5 μg/ml transferrin, about 5 ng/ml selenium, antibioticsas described above, and about 0.5% v/v to about 20% v/v fetal calfserum, as described above. The insulin, transferrin, and selenium aresupplied by Collaborative Research of Lexington, Mass. Other reagentssuitable for use in the present invention, unless otherwise specified,are supplied by Sigma Chemical Co. of St Louis, Mo.

When the cells have grown to confluence (about 3-7 days from the initialculturing of the cells), they are harvested, for example, bytrypsinizing the cell monolayer or by other methods known to thoseskilled in the art, and resuspending the cells in a medium such as MEMsupplemented with about 0.5% v/v to about 20% v/v fetal calf serum. Aportion of the cells are reseeded into sterile tissue culture flasks,after which time the cells are again grown in an atmosphere of about 5%v/v CO₂ at about 37° C.

While only two methods for the culturing of RPE cells are described,other suitable methods for culturing RPE cells are known in the art.Such methods are also suitable for use in the practice of the presentinvention.

B. Preparation of Retinal Pigmented Epithelium Conditioned Medium(RPE-CM)

PEDF is a secreted protein, and RPE cells secrete PEDF into the mediumin which they are grown. Therefore, a convenient method of producingPEDF is to grow RPE cells in culture and to periodically harvest themedia from the cultures.

A method suitable for use in the present invention for isolating PEDFfrom medium is to allow RPE cells to grow to confluence in a medium suchas DMEM supplemented with about 1 mM sodium pyruvate, about 0.625 mMHepes, about 6 mM L-glutamine, about 1% w/v non-essential amino acids,about 5 μg/ml insulin, about 5 μg/ml transferrin, about 5 ng/mlselenium, antibiotics and/or fungicides as described above, and about0.5% v/v to about 20% v/v fetal calf serum, as described above. Theconfluent cultures of RPE cells are washed extensively with Hank'sbalanced salt solution, or other suitable wash solutions, to removeserum proteins derived from the fetal calf serum present in the mediaused to culture the RPE cells. About 1 ml, per cm² of cell surface area,of serum-free medium such as DMEM supplemented with about 1 mM sodiumpyruvate, about 0.625 mM Hepes, about 6 mM L-glutamine about 1% w/vnon-essential amino acids, about 5 μg/ml insulin, about 5 μg/mltransferrin, about 5 ng/ml selenium, and antibiotics and/or fungicidesas described above, is added to the cultures, and they are incubated inan atmosphere of about 5% v/v CO₂ at about 37° C. for about 1 to about10 days. Alternatively, PEDF can be isolated from serum-containingmedium such as DMEM supplemented with about 1 mM sodium pyruvate, about0.625 mM Hepes, about 6 mM L-glutamine, about 1% w/v non-essential aminoacids, about 5 μg/ml insulin, about 5 μg/ml transferrin, about 5 ng/mlselenium, antibiotics and/or fungicides as described above, and about0.5% v/v to about 20% v/v fetal calf serum as described above.

The medium is then collected by pouring the medium into plasticcentrifuge tubes, and the medium is centrifuged at about 3,000 rpm forabout 10 minutes to remove any free cells and other particulate matterfrom the medium, and filtered to provide an impure PEDF proteinsolution. The medium may be used directly for the purification ortesting of PEDF or it may be stored at −20° C. until required.

C. Isolation of PEDF from Tissue Samples

PEDF may be isolated directly from eyes by opening the eyes and removingthe vitreous body and retina. The retinal pigmented epithelium isscraped off the Bruch's membrane using a disposable cell scraper. Tissuefragments are transferred to a Teflon or glass homogenizer in a solutionsuch as 10 mM phosphate-buffered saline (PBS) and homogenized to breakthe cells. The solution is then centrifuged at about 10,000 rpm forabout 10 minutes and filtered to remove cell debris. The supernatant,which contains PEDF, is collected to provide an impure PEDF proteinsolution. The medium may be used directly for the purification ortesting of PEDF or it may be stored at −20° C. until required.

D. Isolation of PEDF from Bovine Vitreous

The vitreous is a gel like substance which fills the eye cup. Thevitreaous is removed from the eye and solubilized by freezing andthawing. About 20 ml of solubilized vitreous are obtained per bovine eyecup and this volume contains about 16 μl/100 μl of PEDF.

F. Isolation of PEDF from Recombinant Cells

PEDF may also be isolated from recombinant cells which have beenconstructed to express the PEDF gene. The PEDF may be expressed as anintracellular or an extracellular protein.

Intracellular PEDF is isolated by homogenizing the cells in a Dounce orother suitable homogenizer in a buffer, such as PBS, that may containdetergents or other solubilizing agents such as urea or guanidinehydrochloride, and centrifuging at about 10,000 rpm for about 20 minutesto remove cellular debris, to provide an impure PEDF protein fraction.

Extracellular PEDF is isolated by collecting the medium in which cellsexpressing PEDF are grown. This is most conveniently performed in acontinuous centrifugation process, such as with a Sharples centrifuge.The supernatant is collected to provide an impure PEDF protein fraction.The medium may be used directly for the purification or testing of PEDFor it may be stored at −20° C. until required.

2. Purification of PEDF

A. Small Scale Purification of PEDF

i. Ammonium Sulfate Precipitation

An impure PEDF protein fraction may be partially purified by ammoniumsulfate precipitation to provide an ammonium sulfate purified PEDFprotein fraction. Percent ammonium sulfate refers to % saturation ofammonium sulfate at 20° C. and is based on a 100% saturation of 767 g/l.

An impure PEDF protein fraction is brought to about 50% saturation withammonium sulfate by the addition of about 313 g of solid ammoniumsulfate per liter of impure protein fraction at about 20° C. Theammonium sulfate is preferably added slowly to the impure proteinfraction while the solution is stirred, such as with a stir bar on amechanical stirrer. The ammonium sulfate is added slowly to preventlocalized high concentrations of ammonium sulfate that may result inrapid precipitation and denaturation of proteins present in the impureprotein fraction. After all the ammonium sulfate is added, the about-50%ammonium sulfate solution is stirred for about 30 minutes at about 20°C. The about-50% ammonium sulfate solution is then centrifuged at about10,000 g, at about 20° C. for about 20 minutes. The supernatant iscollected for further processing. Alternatively, the about-50% ammoniumsulfate solution may be filtered through filter paper such as Whatman#1, to remove the precipitate. In this case, the filtrate is collectedfor further processing.

The about-50% ammonium sulfate solution is then brought to about 70%saturation by the addition of about 137 g/l of ammonium sulfate. Theammonium sulfate is added slowly, and after all the ammonium sulfate isadded, the about-70% ammonium sulfate solution is stirred for about 30minutes at about 20° C. The about-70% ammonium sulfate solution iscentrifuged or filtered, as described above, and the precipitate iscollected.

The ammonium sulfate precipitate is then redissolved in a buffer such asabout 10 mM phosphate, pH 7, to form a 50-70% ammonium sulfate fraction.The solution is then diafiltered using an Amicon Diaflo ultrafiltrationunit, or dialyzed against a buffer such as about 10 mM phosphate, pH 7,to remove the residual ammonium sulfate from the redissolved 50%-70%ammonium sulfate fraction.

ii. Purification of PEDF by SDS Gel Electrophoresis

A small-scale isolation of PEDF is conducted by SDS-polyacrylamide slabgels. Such polyacrylamide gels are well known in the art, and thepreparation of such gels has been described by Weber and Osborn, J.Biol. Chem. 244 4406 (1969), as modified by Laemmli, Nature 277 680(1970).

Samples of an impure PEDF protein fraction or ammonium sulfate purifiedPEDF protein fraction are dialyzed against a buffer such as about 10 nMsodium phosphate buffer, pH 7.5, lyophilized and resuspended in 62.5 mMTris, pH 6.8, 2% w/v SDS, 10% v/v glycerol, 0.001% w/v bromophenol blue,and 0.1 M 2-mercaptoethanol, wherein the bromophenol blue is a migrationmarker. Additional samples to be loaded on the gel includemolecular-weight standards such as phosphorylase B, bovine serumalbumin, ovalbumin, carbonic anhydrase, soybean trypsin inhibitor andlysozyme (such as supplied by Bio-Rad, catalog #161-0304), and controlsas may be necessary. In cases where conditioned media are used assamples, media which have not been exposed to RPE cells (unconditionedmedia) are included as controls.

The samples are then incubated in a boiling-water bath for about 5minutes and loaded onto SDS-polyacrylamide slab gels. The markers andunconditioned media are loaded into wells on the outside of the gel, andthe impure PEDF protein fractions or the ammonium sulfate purified PEDFprotein fraction are loaded on the remaining inside wells. The samplesare then subjected to electrophoresis. The electrophoresis is continueduntil the bromphenol blue marker has reached the bottom of the gel. Atthe completion of electrophoresis, strips from each outside edge of thegels, which included the markers and a lane each of an impure PEDFprotein fraction and an unconditioned media control, are stained withCoomassie blue. The stained protein bands which develop on the stainedstrips are aligned with the unstained portion of the gel to locate theposition of the proteins present in the impure PEDF protein fraction orthe ammonium sulfate purified PEDF protein fraction but absent fromcontrol media.

A PEDF protein doublet, with an apparent molecular weight of about50,000 to about 55,000, unique to the impure PEDF protein fraction, isexcised and the proteins electro-eluted, by methods known in the art,from the unstained portion of the gel. The eluant is centrifuged toremove gel fragments and the supernatant dialyzed against 10 mMphosphate-buffered saline (145 mM NaCl, 8.1 mM Na₂HPO₄, and 1.9 mMNAH₂PO₄.H₂O) to provide an SDS-polyacrylamide purified PEDF proteinfraction.

iii. Purification of PEDF Using BioRad PREP CELL

Large quantities of PEDF can be purified by using BioRad PREP CELL whichis an electrophoresis unit containing a tube gel. Samples are loadedonto the top of the tube gel and fractions are collected from thebottom. Individual fractions are examined by SDS gel electrophoresis fora 50,000 molecular weight protein. Fractions containing a 50,000molecular weight protein are collected and pooled.

iv. Purification of PEDF by Cation-Exchange HPLC

An impure PEDF protein fraction or an ammonium sulfate purified PEDFprotein fraction is dialyzed against water or a buffer such as about 10mM phosphate buffer, pH 7.2, or other suitable buffer, to remove mediaand salts from the impure protein sample. PEDF may be purified bycation-exchange HPLC using a column chromatography medium, such as thatsupplied under the trade name “Brownlee Aquapore CX-300” by WesternAnalytical, Temecula, Calif., packed into a column, such as a 4.6×30 mmcolumn, or other suitable cation-exchange HPLC chromatography medium.

The chromatography medium is equilibrated with a buffer such as about 10mM phosphate, pH 7.2. The dialyzed impure PEDF protein fraction orammonium sulfate purified PEDF protein fraction is loaded onto thechromatography medium, and the chromatography medium with PEDF bound toit is washed with a buffer such as about 10 mM phosphate, pH 7.2, untilall unbound proteins are washed from the chromatography medium. PEDF iseluted from the chromatograph medium with a linear salt gradient fromabout 0.0 to about 0.5 M NaCl. PEDF elutes as a single peak with a NaClconcentration of about 0.25 M.

The eluted PEDF is concentrated by lyophilization and resolubilized inwater or a buffer such as about 10 mM phosphate buffer, pH 7.2, or othersuitable buffer, to form a cation-exchange HPLC purified PEDF proteinfraction.

iv. Purification of PEDF by Reverse-Phase HPLC

An impure PEDF protein fraction or ammonium sulfate purified PEDFprotein fraction is dialyzed against a buffer such as about 10 mMphosphate buffer, pH 7.2, or other suitable buffer, to remove media andsalts from the impure protein sample. PEDF may be purified byreverse-phase HPLC using a column chromatography medium such as achromatography medium supplied under the trade name “Vydac C8” by TheSeparation Group of Hesperia, Calif., packed into a column such as a4.6×250 mm column or other suitable reverse-phase HPLC chromatographymedium.

The chromatography medium is equilibrated with a solution such as about0.1% v/v trifluoroacetic acid (TFA). The dialyzed impure PEDF proteinfraction or ammonium sulfate purified PEDF protein fraction is loadedonto the chromatography medium, and the chromatography medium with PEDFbound to it is washed with an eluant such as 0.1% v/v TFA, until allunbound proteins are washed from the chromatography medium. PEDF iseluted from the chromatograph medium with a linear gradient from about0.1% v/v TFA in water to about 95% v/v acetonitrile (CH₃CN), 0.1% v/vTFA, 5% v/v H₂O. PEDF elutes as a single peak with a CH₃CN concentrationof about 70% v/v.

The eluted PEDF is concentrated by lyophilization and resolubilized inwater or a buffer such as about 10 mM phosphate buffer, pH 7.2, or othersuitable buffer, to form a reverse-phase HPLC purified PEDF proteinfraction.

V. Size-Exclusion HPLC

An impure PEDF protein fraction, an ammonium sulfate purified PEDFprotein fraction, a cation-exchange HPLC purified PEDF protein fraction,or a reverse-phase HPLC purified PEDF protein fraction may be purifiedby size-exclusion chromatograph using a chromatography medium, such asthat supplied under the trade name “Bio-Rad TSK-250” by Bio-Rad ofRichmond, Calif., packed into a column such as a 7.5×300 mm column. Theimpure PEDF protein fraction, the ammonium sulfate purified PEDF proteinfraction, the cation-exchange HPLC purified PEDF protein fraction, orthe reverse-phase HPLC purified PEDF protein fraction is loaded onto thesize-exclusion chromatography medium, which has been equilibrated with abuffer such as 0.02 M Tris-HCl, pH 7.0, 0.6 M NaCl. PEDF-containingfractions are collected and dialyzed against a buffer such as about 10mM phosphate buffer, pH 7.2 to provide a size-exclusion purified PEDFprotein fraction.

vi. Heparin Chromatography

An impure PEDF protein fraction or an ammonium sulfate purified PEDFprotein fraction may also be purified by heparin chromatography. Aheparin chromatography medium such as heparin agarose, supplied by SigmaChemical Co. of St Louis, Mo. (Cat. No. H-5380), is equilibrated with abuffer such as about 10 mM Tris-HCl, pH 7.5.

An impure PEDF protein fraction or an ammonium sulfate purified PEDFprotein fraction is dialyzed against a buffer such as about 10 mM Tris,pH 7.5, to remove any salts or media from the samples, and the dialyzedPEDF solution is applied to the equilibrated heparin agarose. After thePEDF solution has been applied to the heparin agarose, the heparinagarose is washed with a buffer such as about 10 mM Tris-HCl, pH 7.5,until all unbound proteins are eluted from the heparin agarose. PEDF isthen eluted from the heparin agarose with a buffer such as about 10 mMTris-HCl, pH 7.5, 0.5 M NaCl.

The eluate containing PEDF is then diafiltered in an Amicon Diafloultrafiltration unit, or is dialyzed against a buffer such as about 10mM Tris-HCl, pH 7.5, to remove NaCl present in the eluate, therebyproviding a heparin purified PEDF protein fraction.

The above-described purification procedures may use an impure PEDFprotein fraction or an ammonium sulfate purified PEDF protein fractionas the starting material for the subsequent column purification.Alternatively, a protein fraction which has already been purified by oneor more of the described chromatography steps could also be used.Therefore, the purification procedures may be used alone or incombination with each other or with other purification techniques knownin the art to produce a PEDF protein fraction of the desired purity.

B. Large-Scale Preparation of PEDF

i. Ammonium Sulfate Precipitation

Large-scale purification of PEDF may be prepared by ammonium sulfateprecipitation to provide an ammonium sulfate purified PEDF proteinfraction.

An impure PEDF protein fraction is brought to about 50% saturation withammonium sulfate by the addition of about 313 g of solid ammoniumsulfate per liter of impure PEDF protein fraction. The ammonium sulfateis preferably added slowly to the impure protein fraction while thesolution is stirred, such as with a stir bar on a mechanical stirrer.The ammonium sulfate is added slowly to prevent localized highconcentrations of ammonium sulfate which may result in rapidprecipitation, and denaturation of proteins present in the impureprotein fraction. After all the ammonium sulfate is added, the 50%ammonium sulfate solution is stirred for about 30 minutes at 20° C. The50% ammonium sulfate solution is then centrifuged at about 10,000 g, at20° C. for about 20 minutes. The supernatant is collected for furtherprocessing. Alternatively, the 50% ammonium sulfate solution may befiltered through filter paper, such as Whatman #1, to remove theprecipitate. The filtrate is collected for further processing.

The 50% ammonium sulfate solution is then brought to about 70%saturation by the addition of about 137 g/l of ammonium sulfate. Theammonium sulfate is added slowly, and after all the ammonium sulfate isadded, the 70% ammonium sulfate solution is stirred for about 30 minutesat 20° C. The 70% ammonium sulfate solution is centrifuged or filtered,as described above, and the precipitate is collected.

The ammonium sulfate precipitate is then redissolved in a buffer such asabout 10 mM phosphate, pH 7, to form a 50-70% ammonium sulphatefraction, then diafiltered using an Amicon Diaflo ultrafiltration unit,or dialyzed against about 10 mM sodium phosphate, pH 7, to remove theammonium sulfate from the redissolved 50-70% fraction to provide anammonium sulfate purified PEDF protein fraction.

ii. Anion-Exchange Chromatography

For use, an anion-exchange chromatography medium such as DEAE celluloseis equilibrated with a buffer such as about 10 mM Tris, pH 7.5. The PEDFis applied to the anion-exchange chromatography medium, and the mediumis washed with a buffer such as about 10 mM Tris, pH 7.5, until allunbound proteins are eluted from the column. After all the unboundproteins are eluted, PEDF is eluted with a linear salt gradient fromabout 0 to about 1 M NaCl in a buffer such as about 10 mM Tris-HCl, pH7.5. The fractions containing PEDF are collected and pooled. Thesefractions are then diafiltered or dialyzed against a buffer such asabout 10 mM Tris-HCl, pH 7.5, to remove NaCl, thereby providing ananion-exchange chromatography purified PEDF protein fraction.

Anion-exchange chromatography may be conducted by either batch or columnchromatography techniques.

iii. Heparin Chromatography

Heparin chromatography may also be used for the large-scale purificationof PEDF. A heparin chromatography medium such as heparin agarose,supplied by Sigma Chemical Co. (Cat. No. H-5380), is equilibrated with abuffer such as about 10 mM Tris-HCl, pH 7.5.

An impure PEDF protein fraction or an ammonium sulfate purified PEDFprotein fraction is dialyzed against a buffer such as about 10 mMTris-HCl, pH 7.5, and then applied to the heparin agarose. After thePEDF solution has been applied to the heparin agarose, the heparinagarose is washed with a buffer such as about 10 mM Tris-HCl, pH 7.5,until all unbound proteins are eluted from the heparin agarose. PEDF isthen eluted from the heparin agarose with a buffer such as about 10 mMTris-HCl, pH 7.5, 0.5 M NaCl.

The eluant containing PEDF is collected and diafiltered in an AmiconDiaflo ultrafiltration unit, or dialyzed against a buffer such as about10 mM Tris-HCl, pH 7.5, to remove the NaCl present in the eluate, toprovide a heparin purified PEDF protein fraction.

The above-described purification procedures may use an impure PEDFprotein fraction or an ammonium sulfate purified PEDF protein fractionas the starting material for the subsequent column purification.Alternatively, a protein fraction which has already been purified by oneor more of the described chromatography steps could also be used.Therefore, the purification procedures may be used alone or incombination with each other or with other purification techniques knownin the art to produce a PEDF protein fraction of the desired purity.

3. PEDF Assays

A. SDS Gel Electrophoresis

PEDF may be identified by SDS-polyacrylamide gel electrophoresis on, forexample, 7.5%, 10%, 12.5% w/v SDS-polyacrylamide gels. The preparationof such gels has been described by Weber and Osborn J. Biol. Chem. 2444406 (1969), as modified by Laemmli, Nature 277 680 (1970), and thepreparation and use of such gels are well known in the art.

The PEDF protein samples are mixed with about 5 μl of 62.5 mM Tris, pH6.8, 12% w/v SDS, 0.001% w/v bromophenol blue, 10% v/v glycerol, and 0.1M 2-mercaptoethanol, wherein the bromophenol blue is a marker.Molecular-weight marker samples which include molecular-weight standardssuch as phosphorylase B, bovine serum albumin, ovalbumin, carbonicanhydrase, soybean trypsin inhibitor and lysozyme (such as supplied byBio-Rad, catalog #161-0304) are included on the gels as controls. ThePEDF protein samples and the molecular-weight standards are boiled forabout 5 minutes and loaded onto separate wells on the SDS-polyacrylamideslab gels. The samples are then subjected to electrophoresis until thebromphenol blue has migrated to the bottom of the gel. At the completionof electrophoresis, the gel is silver-stained, stained with Coomassieblue, or stained by other suitable protein staining methods. Themolecular weight of proteins in the PEDF sample is then compared to themolecular-weight standards.

Alternatively, larger quantities of PEDF can be collected from SDS-PAGEtube gels using BioRad PREP CELL equipment and a fraction collector.

PEDF migrates as a protein doublet, with an apparent molecular weight offrom about 50,000 to about 55,000 on SDS polyacrylamide gels.

B. Neuronal Inductivity

PEDF activity in protein samples may be assayed by its neuronalinductivity. Various concentrations of PEDF are added to cultures ofcells such as Y79 retinoblastoma (RB) cells, supplied by the AmericanType Culture Collection, Access No. HTB 18, of Rockville, Md.

The Y79 RB cells are grown in suspension culture. The cells areharvested by centrifugation for about 5 minutes at about 900 rpm at roomtemperature and resuspended in a serum-free medium such as Dulbecco'smodified Eagle's medium supplemented with 5 ug/ml insulin, 5 ug/mltransferrin, 5 ug/ml selenous acid, and 876.6 ug/ml L-glutamine(serum-free medium), which has previously been warmed to about 37° C.The collected cells are resuspended at a concentration of about 10⁶cells/ml in serum-free medium. About 50 to about 500 ng/ml of PEDF isadded to about 25 ml aliquots of the cells. The cells are incubated forabout 7 days at about 37° C., then attached to poly-D-lysine-coatedflasks or glass coverslips. The poly-D-lysine-coated flasks are preparedby coating with a solution containing about 200 ug/ml poly-D-lysine(such as that supplied by Sigma, Catalog #P7405) for about 1-24 hours,followed by rinsing with water and serum-free medium. Other cells may beused and it will be clear to one skilled in the art that testing othercells is a routine matter of following the methods set out above withthe desired cell line.

i. Cell Analyses

Morphology of attached cells is monitored daily by phase contrastmicroscopy of living cells using a microscope such as an invertedDiaphot TMD microscope, supplied by Nikon of Tokyo, Japan, or bydifferential interference contrast microscopy of cells, fixed with afixative such as about 4% v/v paraformaldehyde in 0.1 M sodiumcacodylate buffer, using a microscope, such as a BHS-BH2 microscope,supplied by Olympus of Tokyo, Japan.

Differentiation is assessed by calculating the percentage of cellularaggregates (more than 90% of Y79 cells plated from suspension cultureattach to poly-D-lysine-coated flasks as aggregates containing more than5 cells) in which cells extend processes at day 1, 3, 7 and 11 afterattachment. Experiments are performed in replicates of 3 and arerepeated twice.

Expression of neuron-specific enolase (NSE) and neurofilament 200,000molecular weight protein subunit (NF 200) is monitored byimmunofluorescence and viewed by either epifluorescence microscopy(Olympus BHS-BH2 microscope) or microspectrofluorometry (MSA, FarrandMicroscope Spectrum Analyzer). Quantification is by 1) visual scoring ofintensity of fluorescence at 485 nm excitation as +=weak; ++=moderate;+++=strong; ++++=very intense, and 2) microspectrofluorometric analysis(MSA) readings (μA×100, time constant of about 0.3 seconds; specimensize of approximately 2 mm or about 100 cells/aggregate; target size ofabout 15 μm or approximately that of 1 cell).

The presence of PEDF in the protein sample results in the Y79 RB cells,extending neurite-like processes. At a concentration of about 500 ng/ml,about 70% of the cells extend neurite-like processes.

ii. Differentiation

Cells were cultured as described above. The cells were then monitoreddaily by phase-contrast microscopy of living cells (Olympus IMT-2) andby differential interference contrast microscopy (Olympus BHS) of cellsfixed with a fixative such as about 4% v/v paraformaldehyde. Thepercentage of differentiating cells is estimated by calculating thenumber of cellular aggregates containing five or more cells exhibitingneurite outgrowths following 8-10 days of culture on a poly-D-lysinesubstratum.

With about 50 to about 500 ng/ml PEDF, approximately 80% of the cellsundergo morphological differentiation within about 3 days.

4. Characterization of the PEDF Protein

A. Isolation of PEDF Peptides

Purified PEDF, about 500 μg, is concentrated using Centricon 10microconcentrators (Amicon, Danvers, Mass.), and then diluted in asuitable digestion buffer such as about 25 mM Tris, pH 8.5, 1 mM EDTA.To the protein sample is added a proteolytic enzyme, such asendoproteinase Lys-C, supplied by Boehringer-Mannheim of Indianapolis,Ind. The PEDF/proteinase mixture is incubated for about 18 hours atabout 30° C., or until the reaction has gone to completion, i.e., untilall the PEDF is completely digested by the proteinase. Alternatively,the protein may be digested with trypsin in a buffer comprising about 10mM PBS or by using other proteinases known in the art.

The resulting PEDF polypeptide fragments are separated by using aseparation system such as HPLC on a Vydac C8 reverse-phase column. A4.6×250 mm column is suitable for use in the present invention. Thecolumn is equilibrated with about 0.1% v/v TFA in water. Thepolypeptides are eluted with about 90% v/v CH₃CN, 0.1% v/v TFA and 5%v/v H₂O.

Polypeptides eluted from the column which are well separated from otherpolypeptides are collected and subjected to protein sequencing analysis.

B. Protein Sequencing Analysis

The purified polypeptide fragments are subjected to amino-acid sequenceanalysis by methods known in the art. Alternatively, the amino-acidsequence analysis is conveniently performed under contract at anamino-acid sequencing facility, such as the Microsequencing Facility ofBeckman Research Institute at the City of Hope in Duarte, Calif.

5. Characterization of the PEDF Gene

The present invention provides, among other things, a nucleic acid whichencodes PEDF. In particular, a cDNA sequence is provided for human PEDFas set forth in SEQ ID NO:1, for mouse PEDF as set forth in SEQ ID NO:7and for human PEDF as set forth in SEQ ID NO:8. This cDNA sequence codesfor PEDF, which has the amino acid sequence set forth in SEQ ID NO:2.The cDNA and amino acid sequences are listed in the GenBank® Data Bankunder accession number M76979.

The term “nucleic acid” refers to a polymer of deoxyribonucleic acid(DNA) or ribonucleic acid (RNA), which can be derived from any source,can be single- or double-stranded, and can optionally contain synthetic,non-natural, or altered nucleotides which are capable of beingincorporated into DNA or RNA polymers. The nucleic acid of the presentinvention is preferably a segment of DNA.

The present invention further provides a truncated version of PEDF,which is referred to as PEDF-BH. PEDF-BH comprises the amino acidsequence Met-Asn-Arg-Ile fused to Asp⁴⁴ . . . Pro⁴¹⁸ of PEDF, the aminoterminus of which has been deleted. The truncated protein comprises theamino acid sequence of SEQ ID NO:3. The present invention also providesa nucleic acid which encodes a protein comprising the amino acidsequence of PEDF-BH, i.e., the amino acid sequence of SEQ ID NO:3.

One who is skilled in the art will appreciate that more than one nucleicacid may encode any given protein in view of the degeneracy of thegenetic code and the allowance of exceptions to classical base pairingin the third position of the codon, as given by the so-called “Wobblerules.” Moreover, nucleic acids that include more or less nucleotidescan result in the same or equivalent proteins. Accordingly, it isintended that the present invention encompass all nucleic acids thatencode the amino acid sequences of SEQ ID NO:2 and SEQ ID NO:3, as wellas equivalent proteins. The phrase “equivalent nucleic acids” isintended to encompass all of these nucleic acids.

It also will be appreciated by one skilled in the art that amino acidsequences may be altered without adversely affecting the function of aparticular protein. In fact, some alterations in amino acid sequence mayresult in a protein with improved characteristics. The determination ofwhich amino acids may be altered without adversely affecting thefunction of a protein is well within the ordinary skill in the art.Moreover, proteins that include more or less amino acids can result inproteins that are functionally equivalent. Accordingly, it is intendedthat the present invention encompass all amino acid sequences thatresult in the PEDF and PEDF-BH proteins, proteins that are functionallyequivalent to PEDF and PEDF-BH, and proteins derived therefrom. Thephrase “equivalent proteins” is intended to encompass all of these aminoacid sequences.

Some examples of possible equivalent nucleic acids and equivalentproteins include nucleic acids with substitutions, additions, ordeletions which direct the synthesis of the PEDF and PEDF-BH proteinsand equivalent proteins; nucleic acids with different regulatorysequences that direct the production of the PEDF and PEDF-BH proteins;variants of PEDF-BH which possess different amino acids and/or a numberof amino acids other than four fused to the amino terminal end of theprotein; and PEDF and PEDF-BH proteins with amino acid substitutions,additions, deletions, modifications, and/or posttranslationalmodifications, such as glycosylations, that do not adversely affectactivity.

The present invention also provides a vector which comprises a nucleicacid of SEQ ID NO:1, a nucleic acid which encodes a protein comprisingthe amino acid sequence of SEQ ID NO:2 or an equivalent protein, anucleic acid which encodes a protein comprising the amino acid sequenceof SEQ ID NO:3 or an equivalent protein, and equivalent nucleic acidsthereof.

A. Cloning of the PEDF cDNA

Oligonucleotides are constructed from the sequence derived for theisolated polypeptide of PEDF on an ABI 392 DNA/RNA Synthesizer or bymethods well known in the art.

The oligonucleotides are used as primers for a polymerase chain reaction(PCR) by using a Techne thermal cycler and standard reagents andmethodologies, supplied under the trade name “GeneAMP” byPerkin-Elmer/Cetus of Emeryville, Calif.

A human fetal eye Charon BS CDNA library is screened by PCR techniquesusing a Techne thermal cycler and standard reagents and methodologies.The cDNA fragment generated by the reaction is isolated on a 3% w/vNuSieve 3:1 gel (FMC Biochemicals) using NA-45 DEAE-cellulose paper(Schleicher and Schull) as described by Sambrook et al., 1989 In:Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring Harbor Press,Cold Spring Harbor, N.Y. Briefly, the screening procedure is performedby taking about 1, 5 and 50 μl aliquots of the library and placing themin 600 μl siliconized reaction tubes and then bringing them to a finalvolume of about 74 μl with double-distilled sterile water. The phageparticles are disrupted by incubation at about 70° C. for about 5minutes and then cooling on wet ice.

PCR master mix is made up in a 600 μl reaction tube for 3 reaction tubesas follows:

30 μl of 10× Taq polymerase buffer;

24 μl of dNTP mix; and an appropriate volume of double-distilled wateris added to bring the master mix to a final volume of about 78 μl.

The final solution, therefore, comprises about 192 mM KCl, 38.5 mMTris-HCl, pH 8.3, 51.8 mM MgCl₂, 0.038% w/v gelatin, 0.77 mM of eachdNTP, and 3.8 μl of each oligonucleotide primer. About 26 μl of mastermix is added to each reaction tube. The library aliquots and the mastermix solutions are overlayed with about 100 μl of mineral oil and heatedto about 94° C. for about 5 minutes. The solutions are then equilibratedto the desired primer annealing temperature.

After the solutions have been equilibrated to the desired primerannealing temperature, about 1.5 μl of Taq polymerase is added to thesolution and incubated for about 20 minutes.

Thermal cycling is continued by incubating: at about 72° C. for about 3minutes for primer extension, however, this primer extension time may bevaried if desired; at about 94° C. for about 1 minutes about 20 secondsto denature the extension product from the template; at about 37° C. forabout 2 minutes to anneal the primers to a template; and at about 72° C.for about 3 minutes for primer extension. The “cycle” is the repeatedfor a total of about 25 to about 30 cycles. At the end of the lastcycle, a final primer extension step, of about 7 minutes at 72° C. isadded.

The fragment is labelled with ³²p by random priming using a kit suppliedunder the trade name “Prime-It Random Primer Labeling Kit” supplied byStratagene Inc., La Jolla, Calif. The labelled probe is used to screenabout 200,000 plaque-forming units of the Charon BS cDNA library bymethods well known in the art.

Positive clones are isolated, and the DNA purified, with reagentssupplied under the trade name “Qiagen Maxi” by Qiagen Inc. of StudioCity, Calif.

The inserts within the phage vector are excised with an appropriaterestriction enzyme, circularized with T4 DNA ligase supplied by NewEngland Biolabs of Beverly, Mass. and transformed into competent E. coliSure cells supplied by Stratagene. The cells are then plated onampicillin/5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-gal) plates.

White colonies are selected and mini-prepped using a plasmid miniprepreagent supplied by Qiagen. Purified plasmids are digested to excise theinsert, and the fragments are separated by gel electrophoresis todetermine the size of the inserts. A clone with an appropriately-sizedinsert is then chosen for further investigation.

The cDNA's for mouse and bovine PEDF were obtained following similarprocedures.

B. Cloning of the Genomic DNA Sequence

The cDNA insert is labeled ith α³²P dCTP and used to screen two genomicDNA libraries: a cosmid library constructed from MboI partial digests ofhuman placental DNA (Clonetech) and a human placental genomic libraryconstructed in λ DASH II (Stratagene). Positively hybridizing clones areanalyzed by Southern blot analysis. Two strongly hybridizing fragments:a 7.1 kb BamHI fragment from the cosmid clone and a 7.2 kb Not1 fragmentfrom the λ DASH II clone are selected for subcloning and DNA sequencing.

In an alternative embodiment of the present invention, primers, designedfrom the internal coding region of the PEDF cDNA sequence aresynthesized using an ABI 392 DNA/RNA synthesizer and used as primers inpolymerase chain reaction (PCR) experiments. The primer sequences are asfollows: primer 603:

5′-ACAAGCTGGC AGCGGCTGTC-3′ (SEQ ID NO:9) is located in SEQ ID NO:1 at271-290; primer 604:

5′-CAGAGGTGCC ACAAAGCTGG-3′ (SEQ ID NO:10) is located on the antisensestrand in SEQ ID NO:1 at 570-590; primer 605:

5′-CCAGCTTTGT GGCACCTCTG-3′ (SEQ ID NO:11) is located in SEQ ID NO:1 at570-590; primer 606:

5′-CATCATGGGG ACCCTCACGG-3′ (SEQ ID NO:12) is located on the antisensestrand in SEQ ID NO:1 at 829-848; primer 2213:

5′-AGGATGCAGG CCCTGGTGCT-3′ (SEQ ID NO:13) is located in SEQ ID NO:1 at114-133; primer 2744:

5′-CCTCCTCCAC CAGCGCCCCT-3′ (SEQ ID NO:14) is located on the antisensestrand in SEQ ID NO:1 at 224-244; primer 2238:

5′-ATGTCGGACC CTAAGGCTGT T-3′ (SEQ ID NO:15) is located in SEQ ID NO:1at 846-866; primer 354:

5′-TGGGGACAGT GAGGACCGCC-3′ (SEQ ID NO:16) is located on the antisensestrand in SEQ ID NO:1 at 1043-1062. The primer pairs 603:604 amplified asingle 2 kb PCR product (jt108); 605:606 amplified a single 3.3 kb PCRproduct (jt109); 2213:2744 amplified a single 2.3 kb PCR product (jt115)and 2238:354 amplified a single 1.5 kb PCR product (jt116).

In an alternative embodiment of the present invention, two sets ofprimers JT10-UP01:JT10-DP01 corresponding to bases 6536-6559 of jt106genomic sequence and 1590:1591 corresponding to bases 1-89 of SEQ IDNO:1 are used in PCR reactions to isolate P1 clones (Genome Systems).The primer sequences are as follows: primer JT10-UP01:

5′-GGTGTGCAAA TGTGTGCGCC TTAG-3′ (SEQ ID NO:17) is located in SEQ ID NO:4 at 6536; primer JT10-DP01:

5′-GGGAGCTGCT TTACCTGTGG ATAC-3′ (SEQ ID NO:18) is located in SEQ IDNO:4 at 7175; primer 1590:

5′-GCACGCTGGA TTAGAAGGCA GCAAA-3′ (SEQ ID NO:19) is located is SEQ IDNO:1 at 1-25; and primer 1591:

5′-CCACACCCAG CCTAGTCCC-3′ (SEQ ID NO:20) is located in SEQ ID NO:1 at71-89.

C. DNA Sequence Analysis

Sequence analysis is performed with an automated sequencer or by othertechniques well known in the art.

6. Expression of the PEDF Gene in Recombinant Cells

Commercial or large-scale production of PEDF may be achieved byexpression of the gene, after cloning into an appropriate vector, in asuitable host cell. One such suitable vector/host system is thebaculovirus/insect cell systems.

In one embodiment of the present invention, the PEDF gene is cloned intoa baculovirus transfer vector such as pAC373, described by Lithgow etal., DNA and Cell Biology 10 443-449 (1991); pVL941, described by Ghiasiet al., Virology 185 187-194 (1991); or other such baculovirus transfervectors that are well known by those skilled in the art.

Generally, such vectors comprise the polyhedron promoter of theAutographa californica nuclear polyhedrosis virus (AcNPV), inserted intoa transfer vector such as pAc373, described by Summers and Smith (Amanual for baculovirus vector and insect cell culture procedures, TexasAgric. Exp. Station Bull. No. 1555). Recombinant plasmids are preparedby methods well known in the art, such as those described by Maniatis etal. In: Molecular Cloning: A Laboratory Manual 2nd ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. (1982).

The host cells, such as S. frugiperda (Sf9), are grown in TC100 mediumsupplied by GIBCO, supplemented with 10% v/v fetal calf serum. The Sf9cells are co-transfected with purified infectious AcNPV DNA, about 1 μg,and about 50 μg of the recombinant DNA. Culture supernatants areharvested about 4 days after transfection. Recombinant viruses areidentified by plaque hybridization or radiolabelling of the proteinsproduced.

The above description provides an example of the expression of the PEDFgene in a vector/host expression system. Other expression systems areknown in the art; for example, Saccharomyces cerevisiae has been used inconjunction with a number of vectors such as those described by Silar etal., Gene 104 99-102 (1991), Dietrich et al., Eur. J. Biochem. 201399-407 (1991), or Akiyoshi-Shibata et al., DNA and Cell Biology 10613-612 (1991); recombinant vaccinia virus vectors in HeLa host cellssuch as those described by Nakano et al., Gene 105 173-178 (1991). Anysuch methods or other methods known in the art are suitable for use inthe present invention for expression of the PEDF gene.

The PEDF gene may be expressed as a transported protein where the PEDFis isolated from the medium in which the recombinant expression host isgrown, or may be expressed as an intracellular protein by deleting theleader or other peptides, in which case the PEDF is isolated from thehost cells. The PEDF so isolated is then purified by the methodsdescribed above.

In another embodiment of the present invention the vector πFS 17, ATCCDeposit No. PTA-2753, which comprises the nucleic acid of SEQ ID NO:1,and the vector pEV-BH which comprises a nucleic acid which encodes aprotein comprising the amino acid sequence of SEQ ID NO:3. It will beappreciated by those skilled in the art that the cDNA inserts describedcan be present in alternative vectors. For example, inserts can be invectors of different nature, such as phages, viral capsids, plasmids,cosmids, phagemids, YACs, or even attached to the outside of a phage orviral capsid. The vectors can differ in host range, stability,replication, and maintenance. Moreover, the vectors can differ in thetypes of control exerted over cloned inserts. For example, vectors canplace cloned inserts under the control of a different promoter,enhancer, or ribosome binding site, or even organize it as part of atransposon or mobile genetic element.

The present invention also provides a host cell into which a vector,which comprises a nucleic acid of SEQ ID NO:1, a nucleic acid whichencodes a protein comprising the amino acid sequence of SEQ ID NO:2 oran equivalent protein, a nucleic acid which encodes a protein comprisingthe amino acid of SEQ ID NO:3 or an equivalent protein, or an equivalentnucleic acid thereof, has been introduced. In particular, the host cellmay have the vector πFS17, which comprises the nucleic acid of SEQ IDNO:1, or the vector pEV-BH, which comprises a nucleic acid which encodesa protein comprising the amino acid sequence of SEQ ID NO:3.

The vectors of the present invention can be introduced into any suitablehost cell, whether eukaryotic or prokaryotic. These host cells maydiffer in their preferred conditions for growth, their nutritiverequirements, and their sensitivity to environmental agents. Anyappropriate means of introducing the vectors into the host cells may beemployed. In the case of prokaryotic cells, vector introduction may beaccomplished, for example, by electroporation, transformation,transduction, conjugation, or mobilization. For eukaryotic cells,vectors may be introduced through the use of, for example,electroporation, transfection, infection, DNA coated microprojectiles,or protoplast fusion.

The form of the introduced nucleic acid may vary with the method used tointroduce the vector into a host cell. For example, the nucleic acid maybe closed circular, nicked, or linearized, depending upon whether thevector is to be maintained as an autonomously replicating element,integrated as provirus or prophage, transiently transfected, transientlyinfected as with a replication-disabled virus or phage, or stablyintroduced through single or double crossover recombination events.

The present invention also provides a method of producing PEDF, PEDF-BH,and equivalent proteins, which method comprises expressing the proteinin a host cell. For example, a host cell into which has been introduceda vector which comprises a nucleic acid of SEQ ID NO:1, a nucleic acidwhich encodes a protein comprising the amino acid sequence of SEQ IDNO:2 or an equivalent protein, a nucleic acid which encodes a proteincomprising the amino acid of SEQ ID NO:3 or an equivalent protein, or anequivalent nucleic acid thereof, may be cultured under suitableconditions to produce the desired protein. In particular, a host cellinto which has been introduced the vector πFS17, which comprises thenucleic acid of SEQ ID NO:1, or the vector pEV-BH, which comprises anucleic acid which encodes a protein comprising the amino acid sequenceof SEQ ID NO:3, may be cultured under suitable conditions to produce theproteins comprising the amino acid sequences of SEQ ID NO:2 and SEQ IDNO:3, respectively.

The present invention also provides recombinantly produced PEDF,PEDF-BH, and equivalent proteins, which have been produced in accordancewith the aforementioned present inventive method of culturing anappropriate host cell to produce the desired protein. The production ofa protein such as PEDF by recombinant means enables the obtention oflarge quantities of the protein in a highly purified state, free fromany disease-causing agents which may accompany the protein isolated orpurified from a naturally occurring source organism, and obviates theneed to use, for example, fetal tissue as a source for such a protein.

The provision of cDNA sequences in the present invention also enablesthe obtention of human genomic DNA sequences that encode PEDF or thathave substantial sequence homology to PEDF-BH.

The obtention of genomic DNA sequences from cDNA sequences can beaccomplished using standard techniques.

Recombinant PEDF, PEDF-BH, and equivalent proteins may be supplied asactive agents to cells by a variety of means, including, for example,the introduction of nucleic acids, such as DNA or RNA, which encode theprotein and may be accordingly transcribed and/or translated within thehost cell, the addition of exogenous protein, and other suitable meansof administration as are known to those skilled in the art. In whateverform in which supplied, the active agent can be used either alone or incombination with other active agents, using pharmaceutical compositionsand formulations of the active agent which are appropriate to the methodof administration. Pharmaceutically acceptable excipients, i.e.,vehicles, adjuvants, carriers or diluents, are well-known to those whoare skilled in the art, and are readily available. The choice ofexcipient will be determined in part by the particular compound, as wellas by the particular method used to administer the compound.Accordingly, there is a wide variety of suitable formulations which canbe prepared in the context of the present invention. However,pharmaceutically acceptable excipients not altering the neurotrophicactivity of the recombinant protein are preferred.

7. Treatment with PEDF

A. Treatment of Tumors

PEDF is administered, alone or in conjunction with other clinical (suchas surgical) procedures. PEDF is administered by intravitreal,subretinal, intravenous or intramuscular injection or injected orapplied at sites of tumor growth. The PEDF may be administered alone orin conjunction with compounds, such as polylactic acid, which facilitatea slow, “time release” of the PEDF. Typically, about 0.01 to about 10 μgof PEDF at a concentration of about 1 to about 100 μg/ml in aphysiologic saline solution or other buffered solution is administeredper dose, and about 0 to about 10 doses are administered per day. Whentime-release compounds are included in the composition, they are used ata concentration of about 1 to about 100 μg/ml. Treatment is continueduntil cessation of tumor growth and/or tumor regression is observed.

Treatment with PEDF is effective for retinal tumors such asretinoblastoma, other neuronal tumors such as neuroblastoma, or tumorsof non-neuronal origin. The treatment results in a cessation orreduction in the rate of cell division and a concomitant reduction inthe rate of tumor growth, which in turn results in tumor regression.

B. Neurotrophic Treatment of Ocular Disease

PEDF is administered, alone or in conjunction with other clinical (suchas surgical) procedures. PEDF is administered by intravitreal orsubretinal injection. The PEDF may be administered alone or inconjunction with compounds such as polylactic acid which facilitate aslow, “time release” of the PEDF. Typically, about 0.01 to about 10 μgof PEDF at a concentration of about 1 to about 100 μg/ml in aphysiologic saline solution or other buffered solution is administeredper dose, and about 0 to about 10 doses are administered per day. Whentime-release compounds are included in the composition, they are used ata concentration of about 1 to about 100 μg/ml. Treatment is continueduntil the progress of the pathology is halted and/or reversed.

Treatment with PEDF is directed at diseases of the neural retina,retinal pigmented epithelium, and other ocular tissue. Treatment resultsin enhanced survival and well-being of the photoreceptors and otherocular cells, prolonging their functional life span and delaying theonset of impaired vision and ultimate blindness.

C. Neurotrophic Treatment of Neuronal Cell Pathology

PEDF is administered, alone or in conjunction with other clinical (suchas surgical) procedures. PEDF is administered by intravenous orintramuscular injection or application or injection at the site ofneuronal cell pathology. The PEDF may be administered alone or inconjunction with compounds such as polylactic acid which facilitate aslow, “time release” of the PEDF. Typically, about 0.01 to about 10 μgof PEDF at a concentration of about 1 to about 100 μg/ml in aphysiologic saline solution or other buffered solution is administeredper dose, and about 0 to about 10 doses are administered per day. Whentime-release compounds are included in the composition, they are used ata concentration of about 1 to about 100 μg/ml. Treatment is continueduntil nerve regeneration is completed.

Treatment with PEDF is directed at injuries to nerves and pathologies ofcells. Treatment results in enhanced survival of nerve cells andpromotion of neurite outgrowth and nerve regeneration.

D. Treatment of Conditions Related to Serine Proteinases

PEDF is a member of the serine proteinase inhibitor family of proteins.As such, it is effective in the treatment of conditions caused by serineproteinases or where a serine proteinase inhibitor would beadvantageous. Serine proteinases include, but are not limited to,chymotrypsin, trypsin, subtilisin, elastin, thrombin, and plasmin.Therefore, PEDF is useful as: an anti-coagulant, an anti-thrombotic, ananti-microbial, an anti-fungal, an anti-parasitic, and a contraceptive;in cosmetic preparations as a proteinase inhibitor; as a weight-gainpromoter; in treatments for elastosis, vascular disorders involvingfibrinoid formation, coagulation disorders, arteriosclerosis, ischemia,arthroses diabetes, emphysema, arthritis, septic shock, lung diseases,excessive complement activation, ulcers, ulcerative colitis,pancreatitis, psoriasis, fibrinolytic disease, arthropathy, boneresorption, hypertension, congestive heart failure, cirrhosis, orallergy caused by proteases, as a dermatological agent for skindiscoloration and age-related wrinkling.

For use as an anti-coagulant, an anti-thrombotic, an anti-microbial, ananti-parasitic, or a contraceptive, or in treatment of elastosis,vascular disorders involving fibrinoid formation, coagulation disorders,arteriosclerosis, ischemia, arthroses diabetes, emphysema, arthritis,septic shock, lung diseases, excessive complement activation,pancreatitis, psoriasis, fibrinolytic disease, arthropathy, boneresorption, hypertension, congestive heart failure, cirrhosis, orallergy caused by proteases, PEDF is administered by intravenous orintramuscular injection or site-directed injection or application. ThePEDF may be administered alone or in conjunction with compounds such aspolylactic acid which facilitate a slow “time release” of the PEDF.Typically, about 0.01 to about 10 μg of PEDF at a concentration of about1 to about 100 μg/ml in a physiologic saline solution or other bufferedsolution are administered per dose, and about 0 to about 10 doses areadministered per day. When time-release compounds are included in thecomposition, they are used at a concentration of about 1 to about 100μg/ml. Treatment is continued until progress of the pathology is haltedand/or reversed.

Treatment with PEDF is directed at injuries to nerves. Treatment resultsin promotion of neurite outgrowth and nerve regeneration.

For use in cosmetic preparations as a proteinase inhibitor, arthritis orelastosis PEDF is added to the preparations to a concentration of about0.01 to about 100 μg/ml.

For use in treatment as a weight-gain promoter, ulcers, ulcerativecolitis, or pancreatitis, PEDF may be administered orally at aconcentration of about 0.01 to about 10 μg per kg of body weight perday.

For use as a treatment for conditions such as psoriasis, PEDF may beadministered topically at a concentration of about 0.01 to about 100μg/ml, formulated in a suitable carrier.

EXAMPLE 1 Establishment of RPE Cell Cultures

RPE cells were harvested from post-mortem human eyes, as described byPfeffer et al., J. Cell. Physiol. 117 333-341 (1983). The cells weregrown in 75 cm flasks with Eagles's minimal essential mediumsupplemented with 15% v/v fetal calf serum and 5% v/v CO₂ at 37° C.Cells were grown to confluence, harvested by trypsinizing the cellmonolayer, and resuspended in MEM supplemented with 15% v/v fetal calfserum. A portion of the cells (about 5%) were reseeded into new 75 cm,where the cells were again grown in 5% v/v CO₂ at 37° C.

EXAMPLE 2 Preparation of RPE-Conditioned Medium

Confluent cultures of RPE cells were washed extensively with HBSS,before conditioning, to remove serum proteins. Twenty-five ml ofserum-free MEM was added, and the cultures were incubated with 5% v/vCO₂ at 37° C. for about 48 hours. The conditioned medium was thencollected and centrifuged at 1,000 rpm at room temperature, to removeany free cells and other particulate matter, to provide an impure PEDFprotein fraction.

EXAMPLE 3 Purification of PEDF by SDS Gel Electrophoresis

Proteins contained in human fetal RPE-conditioned medium, prepared asdescribed in Example 2, and in control non-conditioned medium wereanalyzed on an SDS-polyacrylamide slab gel.

The conditioned and non-conditioned media samples were mixed with anelectrophoresis sample buffer (62.5 mM Tris, pH 6.8, 2% w/v SDS, 10% v/vglycerol, 0.001% w/v bromophenol blue, and 0.1 M 2-mercaptoethanol).Molecular-weight markers, such as phosphorylase B, bovine serum albumin,ovalbumin, carbonic anhydrase, soybean trypsin inhibitor and lysozyme(such as that supplied by Bio-Rad), were also mixed with anelectrophoresis sample buffer. All the samples were denatured at 100°C., in a boiling water bath, for 5 minutes and loaded onto a SDScontaining 7.5% w/v polyacrylamide gel. The electrophoresis wasconducted at 20 mA until the bromphenol blue marker dye migrated to thebottom of the gel.

At the completion of electrophoresis, strips from each outside edge ofthe gels, which included the markers and a lane each of conditioned andnon-conditioned media, were stained with Coomassie blue. The stainedprotein bands which developed on the stained strips were used to alignwith the unstained portion of the gel, to locate the proteins present inconditioned medium but absent from non-conditioned medium.

The about 50,000 to about 55,000 molecular-weight PEDF protein doublet,unique to RPE-CM, were excised, and the proteins were electro-elutedfrom the unstained portion of the gel. A strip of the same gel excisedfrom the region containing bovine serum albumin was treated similarly toserve as a control. The eluant was centrifuged to remove gel fragments,and the supernatant was dialyzed and analyzed by gel electrophoresis toassess its purity.

The electrophoretic patterns of human fetal RPE-CM and non-conditionedcontrol medium show the presence of the prominent about 50,000 to about55,000 molecular-weight PEDF doublet unique to the conditioned medium.

EXAMPLE 4 Small Scale Ammonium Sulfate Precipitation

An impure PEDF protein fraction, prepared as described in Example 2, wasdivided into six 10 ml aliquots. One of the aliquots was brought to 40%saturation, at 20° C., by the addition of 2.42 g of ammonium sulfate;another aliquot was brought to 50% saturation, at 20° C., by theaddition of 3.14 g of ammonium sulfate; another aliquot was brought to60% saturation, at 20° C., by the addition of 3.90 g of ammoniumsulfate; another aliquot was brought to 70% saturation, at 20° C., bythe addition of 4.72 g of ammonium sulfate; another aliquot was broughtto 80% saturation, at 20° C., by the addition of 5.61 g of ammoniumsulfate; and the final aliquot was brought to 90% saturation, at 20° C.,by the addition of 6.57 g of ammonium sulfate. The aliquots were mixeduntil the ammonium sulfate was completely dissolved. The precipitateswhich formed in each of the tubes were collected by centrifugation at10,000 rpm for 20 minutes. The precipitates were each resuspended in 10mM sodium phosphate buffer, pH 7.5, and dialyzed against 2 l of 10 mMsodium phosphate buffer, pH 7.5, for 24 hours.

The samples were then collected and subjected to SDS-polyacrylamide gelelectrophoresis on a 10% w/v SDS-polyacrylamide gel, as described inExample 3.

TABLE I % Saturation Relative concentration Ammonium Sulfate of PEDF 40− 50 − 60 + 70 +++ 80 +++ 90 +++

The results indicate that the PEDF precipitates with an ammonium sulfatesaturation of 60% to 70%. The small amount of PEDF in the 50% to 60%sample indicates that some PEDF is present, and that a suitable ammoniumsulfate “cut” is from 50% to 70% saturation. The samples were subjectedto SDS-polyacrylamide gel electrophoresis, as described in Example 3. Itwas estimated that the purity of the PEDF in the 60% to 70% ammoniumsulfate fraction was about 50%.

EXAMPLE 5 Ammonium Sulfate Precipitation

An impure protein fraction, prepared as described in Example 2, wasbrought to 50% saturation with ammonium sulfate by the addition of 313g/l of solid ammonium sulfate. The ammonium sulfate was added slowly tothe impure protein fraction while the solution was stirred with a stirbar on a mechanical stirrer. After all the ammonium sulfate was added,the 50% ammonium sulfate solution was stirred for 30 minutes at 20° C.The 50% ammonium sulfate solution was then centrifuged at 10,000 g, at20° C. for 20 minutes. The supernatant was collected.

The 50% ammonium sulfate solution was then brought to 70% saturation bythe addition of 137 g/l of ammonium sulfate. The ammonium sulfate wasadded slowly, and after all the ammonium sulfate was added, the 70%ammonium sulfate solution was stirred for 30 minutes at 20° C. The 70%ammonium sulfate solution was centrifuged or filtered, as describedabove, and the precipitate was collected.

The ammonium sulfate precipitate was then redissolved in 10 mM sodiumphosphate, pH 7, to form a 50%-70% ammonium sulphate fraction, thendiafiltered using an Amicon Diaflo ultrafiltration unit, or dialyzedagainst 10 mM sodium phosphate, pH 7, to remove the ammonium sulfatefrom the redissolved 50-70% fraction.

The samples were subjected to SDS-polyacrylamide gel electrophoresis, asdescribed in Example 3. It was estimated that the purity of the PEDF inthe 50% to 70% ammonium sulfate fraction was about 50%.

EXAMPLE 6 Purification of PEDF by Cation Exchange HPLC

RPE-CM, prepared as described in Example 2, was dialyzed against 10 mMsodium phosphate buffer, pH 6.5, to remove media and salts from theimpure protein sample. The dialyzed PEDF sample was then loaded onto aBrownlee Aquapore CX-300 HPLC chromatography medium, packed into a4.6×30 mm column. The chromatography medium was previously equilibratedwith 10 mM phosphate, pH 6.5. The dialyzed RPE-CM was loaded onto thechromatography medium, and the chromatography medium, with PEDF, waswashed with 10 mM phosphate, pH 6.5, until all unbound proteins werewashed from the chromatography medium. PEDF was eluted from thechromatography medium with a linear salt gradient from 0.0 to 0.5 M NaClin 10 mM phosphate, pH 6.5. PEDF, eluted with a NaCl concentration of0.25 M.

The eluted PEDF was concentrated by lyophilization and resolubilized in10 mM phosphate, pH 6.5. The eluted PEDF was analyzed by SDS gelelectrophoresis, as described in Example 3. The eluted PEDF wasestimated to be approximately 70% pure.

EXAMPLE 7 Purification of PEDF by Cation-Exchange HPLC

Ammonium sulfate-precipitated RPE-CM, prepared as described in Example5, was dialyzed against 10 mM sodium phosphate buffer, pH 6.5, to removeammonium sulfate. An 80 μl sample of the dialyzed ammoniumsulfate-purified PEDF protein fraction was loaded onto a BrownleeAquapore CX-300 chromatography medium, packed into a 4.6×30 mm column.The chromatography medium was previously equilibrated with 10 mMphosphate, pH 7.2. The PEDF/chromatography medium was washed with 10 mMsodium phosphate buffer, pH 7.2, until all unbound proteins were washedfrom the chromatography medium. PEDF was eluted from the chromatographymedium with a linear salt gradient from 0.0 to 0.5 M NaCl in 10 mMsodium phosphate buffer, pH 7.2. PEDF eluted as a single peak with aNaCl concentration of about 0.25 M.

The eluted PEDF was dialyzed against 10 mM sodium phosphate buffer, pH7.2, lyophilized, and resolubilized in water. The sample was thensubjected to SDS-polyacrylamide gel electrophoresis, as described inExample 3.

The eluted PEDF was estimated to be about 70% pure.

EXAMPLE 8 Purification of PEDF by Reverse-Phase HPLC

Ammonium sulfate-precipitated RPE-CM, prepared as described in Example5, was dialyzed against 10 mM sodium phosphate buffer, pH 6.5, to removeammonium sulfate. An 80 μl PEDF-containing sample was then loaded onto aVydac C8 chromatography medium, packed into a 4.6×250 mm column. Thechromatography medium was previously equilibrated with 0.1% v/v TFA inwater. The PEDF/chromatography medium was washed with 0.1% v/v TFA inwater until all unbound proteins were washed from the chromatographymedium. PEDF was eluted from the chromatography medium with a lineargradient from 0.0 to 95% v/v CH₃CN, 0.1% v/v TFA in water, and 5% v/vH₂O.

The eluted PEDF was dialyzed against 10 mM sodium phosphate buffer, pH7.2, lyophilized, and resolubilized in water. The sample was thensubjected to SDS-polyacrylamide gel electrophoresis, as described inExample 3.

The PEDF eluted from the column was estimated to be about 80% pure.

EXAMPLE 9 Purification of PEDF by Reverse-Phase HPLC

A PEDF-containing sample, partially purified as described in Example 2,was then loaded onto a Vydac C8 chromatography medium, packed into a4.6×250 mm column. The chromatography medium was previously equilibratedwith 0.1% v/v TFA in water. The PEDF/chromatography medium was washedwith 0.1% v/v TFA in water until all unbound proteins were washed fromthe chromatography medium. PEDF was eluted from the chromatographymedium with a linear gradient from 0.0 to 95% v/v CH₃CN in 0.1% v/v TFAand 5% v/v H₂O.

The eluted PEDF was dialyzed against 10 mM sodium phosphate buffer, pH7.2, lyophilized, and resolubilized in water. The sample was thensubjected to SDS-polyacrylamide gel electrophoresis, as described inExample 3.

The PEDF eluted from the column was estimated to be about 60% pure.

EXAMPLE 10 Purification of PEDF by Reverse-Phase HPLC

A PEDF-containing sample, partially purified as described in Example 6,was then loaded onto a Vydac C8 chromatography medium, packed into a4.6×250 mm column. The chromatography medium was previously equilibratedwith 0.1% v/v TFA in water. The PEDF/chromatography medium was washedwith 0.1% v/v TFA in water until all unbound proteins were washed fromthe chromatography medium. PEDF was eluted from the chromatographymedium with a linear gradient from 0.0 to 95% v/v CH₃CN in 0.1% v/v TFAand 5% v/v H₂O.

The eluted PEDF was dialyzed against 10 mM sodium phosphate buffer, pH7.2, lyophilized, and resolubilized in water. The sample was thensubjected to SDS-polyacrylamide gel electrophoresis, as described inExample 3.

The PEDF eluted from the column was estimated to be about 80% pure.

EXAMPLE 11 Purification of PEDF by Reverse-Phase HPLC

A PEDF-containing sample, prepared as described in Example 6, was loadedonto Brownlee RP-300 chromatography medium, packed into a 4.6×250 mmcolumn. The chromatography medium was previously equilibrated with 0.1%v/v TFA in water. The PEDF/chromatography medium was washed with 0.1%v/v TFA in water until all unbound proteins were washed from thechromatography medium. PEDF was eluted from the chromatography mediumwith a linear gradient from 0.0 to 95% v/v CH₃CN in 0.1% v/v TFA and 5%v/v H₂O.

The eluted PEDF was dialyzed against 10 mM sodium phosphate buffer, pH7.2, lyophilized, and resolubilized in water. The sample was thensubjected to SDS-polyacrylamide gel electrophoresis, as described inExample 3.

The PEDF eluted from the column was estimated to be about 90% pure.

EXAMPLE 12 Purification of PEDF by Size-Exclusion Chromatography

Partially-purified PEDF, prepared as described in Example 5 or 6, waspurified further by chromatography on Bio-Rad TSK-250 chromatographymedium, packed into a 7.5×300 mm column. A 40 μl sample of resolubilizedPEDF was loaded onto the size-exclusion chromatography medium, which hadbeen previously equilibrated with 20 mM Tris, pH 7.0, 0.6 M NaCl. PEDFwas eluted with 20 mM Tris, pH 7.0, 0.6 M NaCl.

The eluted PEDF was dialyzed against 10 mM sodium phosphate buffer, pH7.2, lyophilized, and resolubilized in water. The sample was thensubjected to SDS-polyacrylamide gel electrophoresis, as described inExample 3.

The PEDF eluted from the column was estimated to be about 75% pure.

EXAMPLE 13 Anion-Exchange Chromatography

DEAE cellulose is equilibrated with 10 mM Tris, pH 7.5. PEDF, preparedas described in Example 5, is applied to the column, and the column iswashed with 10 mM Tris, pH 7.5, until all unbound proteins are elutedfrom the column. After all the unbound proteins are eluted, the PEDF iseluted with a linear gradient from 0 to 1 M NaCl in 10 mM Tris-HCl, pH7.5. The fractions containing PEDF are collected and pooled. Thesefractions are then diafiltered against 10 mM Tris-HCl, pH 7.5 to removeNaCl.

EXAMPLE 14 Anion-Exchange Chromatography

DEAE cellulose is equilibrated with 10 mM Tris, pH 7.5. PEDF, preparedas described in Example 2, is applied to the column, and the column iswashed with 10 mM Tris, pH 7.5, until all unbound proteins are elutedfrom the column. After all the unbound proteins are eluted, the PEDF iseluted with a linear gradient from 0 to 1 M NaCl in 10 mM Tris-HCl, pH7.5. The fractions containing PEDF are collected and pooled. Thesefractions are then diafiltered against 10 mM Tris-HCl, pH 7.5 to removeNaCl.

EXAMPLE 15 Cation-Exchange Chromatography

Bio-Rex 70 resin (Bio-Rad) cellulose is equilibrated with 10 mMPhosphate, pH 7.2. PEDF, prepared as described in Example 2, is appliedto the column, and the column is washed with 10 mM Phosphate, pH 7.2,until all unbound proteins are eluted from the column. After all theunbound proteins are eluted, the PEDF is eluted with a linear gradientfrom 0 to 1 M NaCl in 10 mM Phosphate, pH 7.2. The fractions containingPEDF are collected and pooled. These fractions are then diafilteredagainst 10 mM Tris-HCl, pH 7.5 to remove NaCl.

EXAMPLE 16 Cation-Exchange Chromatography

Bio-Rex 70 resin (Bio-Rad) cellulose is equilibrated with 10 mM PBS, pH7.2. PEDF, prepared as described in Example 5, is applied to the column,and the column is washed with 10 mM PBS, pH 7.2, until all unboundproteins are eluted from the column. After all the unbound proteins areeluted, the PEDF is eluted with a linear gradient from 0 to 1 M NaCl in10 mM PBS, pH 7.2. The fractions containing PEDF are collected andpooled. These fractions are then diafiltered against 10 mM Tris-HCl, pH7.5 to remove NaCl.

EXAMPLE 17 Heparin Chromatography

Two ml of heparin agarose was packed into a 0.7×10 cm column, washedwith 20 ml of 10 mM Tris-HCl, pH 7.5, 3 M NaCl, then equilibrated with20 ml of 10 mM Tris-HCl, pH 7.5. Twelve ml of a PEDF solution, preparedas described in Example 2, from a 17-year-old human donor, was dialyzedagainst 4 l of 10 mM sodium phosphate buffer, pH 7.5, at 4° C. for 19hours. The volume after dialysis was 19 ml. The dialysate was applied tothe heparin agarose at a flow rate of 0.4 ml/minutes. After the PEDFsolution had been applied to the heparin agarose, the heparin agarosewas washed with 20 ml of 10 mM Tris-HCl, pH 7.5, to remove unboundproteins from the heparin agarose. PEDF was then eluted from the heparinagarose with 12 ml of 10 mM Tris-HCl, pH 7.5, 3 M NaCl.

The eluate containing PEDF was dialyzed against 10 mM Tris-HCl, pH 7.5,to remove the NaCl present in the eluate. The dialyzed eluate was thenlyophilized, redissolved in 10 mM sodium phosphate buffer, pH 7.5, and asample was subjected to 12.5% w/v SDS-polyacrylamide gelelectrophoresis, as described in Example 3.

The PEDF eluate was estimated to be about 60% pure.

EXAMPLE 18 Heparin Chromatography

Two ml of heparin agarose was packed into a 0.7×10 cm column, washedwith 20 ml of 10 mM Tris-HCl, pH 7.5, 3 M NaCl, then equilibrated with20 ml of 10 mM Tris-HCl, pH 7.5. One ml of a PEDF solution, prepared asdescribed in Example 5, was dialyzed against 4 l of 10 mM sodiumphosphate buffer, pH 7.5, at 4° C. for 19 hours. The volume of thedialysate was 1.3 ml. The dialysate was applied to the heparin agaroseat a flow rate of 0.4 ml/minutes. After the PEDF solution had beenapplied to the heparin agarose, the heparin agarose was washed with 20ml of 10 mM Tris-HCl, pH 7.5, to remove unbound proteins from theheparin agarose. The heparin agarose was then successively eluted with12 ml of 10 mM Tris-HCl, pH 7.5, 0.5 M NaCl; 12 ml of 10 mM Tris-HCl, pH7.5, 1 M NaCl; 12 ml of 10 mM Tris-HCl, pH 7.5, 2 M NaCl; and 12 ml of10 mM Tris-HCl, pH 7.5, 3 M NaCl.

Each of the eluates was dialyzed against 10 mM Tris-HCl, pH 7.5, toremove the NaCl present in the eluate. The dialyzed eluates were thenlyophilized and redissolved in 10 mM sodium phosphate buffer, pH 7.5,and a sample of each dialyzed eluate was subjected to 12.5% w/vSDS-polyacrylamide gel electrophoresis, as described in Example 3.

The PEDF eluate was estimated to be about 70% pure.

EXAMPLE 19 Neuronal Inductivity and Differentiation Activity of IsolatedPEDF

Electro-eluted PEDF was prepared as described in Example 3. Tocharacterize the neuronal inductive activity of the isolated PEDF, theoptimal concentration and pre-seeding stimulatory periods weredetermined. Either 1, 2, 4, 6, 8, or 10 μg/ml of electro-eluted PEDF inserum-free DME, supplemented with 1 nM sodium pyruvate, 0.625 mM HEPES,6 mM L-glutamine, 1% w/v non-essential amino acids, 5 μg/ml insulin, 5μg/ml transferrin, and 5 ng/ml selenous acid, was tested. As a control,RPE-CM, diluted with an equal volume of non-conditioned medium (50%RPE-CM), was also used.

The Y79 RB cells were grown in suspension culture. The cells wereharvested by centrifugation for 5 minutes at 900 rpm at room temperatureand resuspended in serum-free DME medium, which had previously beenwarmed to 37° C. The cells were collected by centrifugation andresuspended at a concentration of 106 cells/ml in serum-free DME medium.One, 2, 4, 6, 8, or 10 μg/ml electro-eluted PEDF or 50% RPE-CM wasintroduced into separate Y79 RB cell cultures. The cells were incubatedfor 7 days at 37° C., and were then attached to poly-D-lysine-coatedflasks. The cells were observed daily by phase-contrast microscopy.

The optimal pre-seeding period required for maximal inductive activityof PEDF was assessed by treating Y79 RB cell cultures with 2 μg/ml ofelectro-eluted PEDF for 2, 4, 8, or 16 days prior to seeding onto apoly-D-lysine substratum. In addition, 1 or 2 μg/ml of electro-elutedPEDF was added to attached cultures of cells not previously stimulatedin suspension culture.

Greater than 80% of Y79 cell aggregates treated with 2 μg/mlelectro-eluted PEDF extended arborizing processes within 8-10 days afterattachment to a poly-D-lysine substratum; untreated cells showed onlyminimal signs of neuronal differentiation.

Y79 cells exhibited maximal differentiative response to electro-elutedPEDF at a protein concentration of 2 μg/ml; the response was reduced atconcentrations exceeding 4 μg/ml. The results are shown in Table II.

TABLE II Pre-Attachment Percent Stimulatory Differentiated PEDF (μg/ml)Period (days) Cells 0 7 13 ± 8¹ 1 7 82 ± 10 2 7 80 ± 10 4 7 33 ± 10 6 75 ± 5 8 7 0 10  7 0 1  0² 20 ± 14 2  0² 28 ± 11 1 μg/ml BSA³ 7 8 ± 6(control) ¹Standard error ²PEDF added to non-stimulated attached cells,24 hours post-seeding ³bovine serum albumin

A pre-seeding stimulatory period (in suspension culture) of 8 days, inthe presence of this factor, results in maximal differentiation of Y79cells at day 10 post-attachment. Decreased frequencies ofdifferentiation (less than 50%) are noted with both shorter and longerpre-seeding inductive periods. In addition, cells not previouslystimulated with PEDF prior to attachment, exhibit a low frequency ofneuronal differentiation (approximately 20%) if 1-2 μg/ml electro-elutedprotein is added post-attachment. This response, however, is relativelyslow, i.e., less than 15 days. Control BSA-treated cultures exhibit lessthan 10% differentiation, comparable to cells exposed only tonon-conditioned media.

PEDF induced a high degree of neuronal differentiation in humanretinoblastoma cells. Long ramifying neuritic processes were induced toextend from aggregates of stimulated cells; concomitant increases in theexpression of the neuronal marker molecules, neuron-specific enolase,and the 200,000 molecular weight neurofilament protein were alsoobserved. The expression of the neuronal phenotype in Y79 cells appearsto involve three sequential events: 1) stimulation of cells insuspension culture by PEDF; 2) attachment of stimulated cells to asubstratum; followed by 3) neurite outgrowth of attached, stimulatedcells. Since only 20% differentiation was observed when non-stimulated,attached cultures were treated with PEDF, commitment to neuronaldifferentiation in Y79 cells appears to precede cell attachment andneurite outgrowth and may be initiated during the stimulatory period.

EXAMPLE 20 Comparison of the Effects of RPE-CM from Different Species

Cell cultures were established as described in Example 1, except theeyes from which the cells were derived were either fetal humans, adulthumans, adult rats, fetal rats, or embryonic chickens.

RPE-CM was prepared from each of the cell cultures, as described inExample 2. Fetal human cell cultures which were newly established(short-term cultures) and cultures which had been established for 6months (long-term cultures) were included.

The differentiation activity of the conditioned media was performed asdescribed in Example 19.

The results of experiments monitoring the effects of RPE-CM from variousspecies are summarized in Table III.

TABLE III Source of RPE-Conditioned Medium Aggregates % DifferentiatedHuman (fetal, short-term cultures) 88 ± 3* Human (fetal, long-termcultures) 50 ± 8 Human (adult) 55 ± 9 Rat (adult) 20 ± 11 Rat (fetal) 42± 7 Chicken (embryonic) 86 ± 7 *Standard error

The table summarizes the inductive activity of a variety ofRPE-conditioned media on Y79 cells. Indicated are the percentages of 10days attached aggregates (more than 5 cells/aggregate) exhibitingneurite outgrowth after 7 days' stimulation with 50% v/v RPE-CM from thevarious species. One hundred aggregates were counted from each sample inreplicates of three.

The greatest differentiative response in Y79 cells is induced by humanfetal RPE-CM (short-term cultures) and embryonic chicken RPE-CM, both ofwhich induce neuronal differentiation in greater than 80% of cellularaggregates when used at a 50% v/v concentration. In contrast, decreasedfrequencies (less than 50%) of cellular differentiation are noted forconditioned media from long-term (12-18 months) cultures of human fetalRPE and adult human RPE. Only 40% neuron-like differentiation is inducedby fetal rat RPE-CM and 20% by adult rat RPE-CM.

Conditioned media from human fetal and embryonic chicken RPE cellscontain similar neurotrophic activity, while those from adult cultures(chicken and rat) and long-term cultures of human fetal RPE-CM are lesseffective in promoting the neuronal phenotype in Y79 cells. It ispossible that mature RPE cells no longer secrete this neurotrophicfactor, or they secrete reduced quantities which are less effective inthe culture conditions utilized. The fact that some trophic activity isseen in RPE-conditioned media from other species suggests that PEDF, ora similar factor, is also secreted by RPE cells of other species and maybe generally important to normal retinal development and function.

EXAMPLE 21 Two-Dimensional Gel Electrophoresis

Two-dimensional gel analysis was conducted by the method described byO'Farrell, J. Biol. Chem. 250 4007-4021 (1975) and Jones, J. Exp. Med14b 1251-1279 (1977). Silver-staining was performed using a Bio-Radsilver-stain plus kit. Twenty μg of PEDF, purified by cation-exchangeand size-exclusion HPLC, was loaded onto the IEF tube gel.

Two-dimensional electrophoretic analysis of HPLC-purified PEDF revealsthe presence of four closely-grouped molecular species. The four speciesvary slightly in apparent molecular weight, but all are in the 50,000molecular-weight region of the gel, consistent with the previouslyidentified molecular weight of PEDF. The resolved species also varyslightly in isoelectric point, suggesting slight variations inpost-translational modifications. Amino-acid sequence analysis (seeExample 22), however, indicated the presence of only a singlepolypeptide.

EXAMPLE 22 Isolation of PEDF Specific Peptides and Amino-Acid Sequences

One-hundred-and-eighty μg of purified PEDF (purified as described inExamples 2, 6, and 12) was concentrated using a Centricon 10microconcentrator, supplied by Amicon of Danvers, Mass., and thendiluted in 25 mM Tris, pH 8.5, 1 mM EDTA. To the protein sample wasadded endoproteinase Lys-C. The PEDF/proteinase mixture was incubatedfor about 18 hours at 30° C. to digest the PEDF.

The resulting PEDF polypeptide fragments were separated using by HPLC ona Vydac C8 reverse-phase HPLC packed into a 4.6×250 mm column. Thecolumn was equilibrated with 0.1% v/v TFA in water. The polypeptideswere eluted with 90% v/v CH₃CN, 0.1% v/v TFA in water.

Polypeptides eluted from the column which are well separated from otherpolypeptides were collected and subjected to protein sequencinganalysis. The amino-acid sequence analysis was performed under contractat the Microsequencing Facility of Beckman Research Institute at theCity of Hope.

The amino-acid sequences for the isolated polypeptides were determinedto be:

PEDF-13 (SEQ ID NO:2), residues 226-244) (SEQ ID NO:21): Thr Ser Leu GluAsp Phe Tyr Leu Asp Glu                   5                  10 Glu ArgThr Val Arg Val Pro Met Met                  15 PEDF-14 (SEQ ID NO:2,residues 161-186) (SEQ ID NO:22): Ser Tyr Gly Thr Arg Pro Arg Val LeuThr                   5                  10 Gly Asn Pro Arg Leu Asp LeuGln Glu Ile                  15                  20 Asn Asn Trp Val GlnAla                  25

Sequencing of PEDF-13 yielded 19 residues of unequivocal sequence.PEDF-14 yielded 26 residues, 25 of which were unequivocal. Theidentification of residue 23 in PEDF-14 as tryptophan was not absolutelycertain.

EXAMPLE 23 Isolation of PEDF Specific Peptides and Amino Acid Sequence

PEDF, purified as described in Examples 2, 6, and 12, was reduced andalkylated. The sample was dried, redissolved in 50 μl of CRA buffer (8 Murea, 0.4 M ammonium bicarbonate, pH 8.0), and 5 μl of 45 mM DTT(Calbiochem) was added. After heating at 50° C. for 15 minutes, thesolution was cooled, and 5 μl of 100 mM iodoacetic acid (Sigma ChemicalCo.) was added. After 15 minutes, the solution was diluted to aconcentration of 2 M urea and subjected to trypsin digestion, suppliedby Boehringer-Mannheim, using an enzyme:substrate ratio of 1:25 (wt/wt),for 22 hours at 37° C.

Tryptic peptides were separated by narrow-bore reverse-phase HPLC on aHewlett-Packard 1090 HPLC, equipped with a 1040 diode array detector,using a Vydac 2.1 mm×150 mm C18 column. Buffer A was 0.06% v/vtrifluoroacetic acid/H₂O, and buffer B was 0.055% v/v trifluoroaceticacid/acetonitrile, a gradient of 5% v/v B at 0 minutes, 33% v/v B at 63minutes, 60% v/v B at 95 minutes, and 80% v/v B at 105 minutes, with aflow rate of 150 μl/minutes, was used. Chromatographic data at 210, 277nm and UV spectra from 209 to 321 nm of each peak were obtained.

Samples for amino terminal sequence analysis were applied to a polybrenepre-cycled glass fibre filter and subjected to automated Edmandegradation at the Harvard Microchemical Facility in Boston, Mass., onan ABI model 477A gas-phase protein sequencer using program NORMAL 1.The resulting phenylthiohydantoin amino acid fractions were manuallyidentified using an on-line ABI Model 120A HPLC and Shimadzu CR4Aintegrator.

The sequences of the isolated peptides were determined to be:

(SEQ ID NO:2 residues 54-67) Leu Ala Ala Ala Val Ser Asn Phe Gly Tyr                  5                  10 Asp Leu Tyr Arg (SEQ ID NO:23)(SEQ ID NO:2) residues 107-135) Ala Leu Tyr Tyr Asp Leu Ile Ser Ser Pro                  5                 10 Asp Ile His Gly Thr Tyr Lys GluLeu Leu                 15                  20 Asp Thr Val Thr Ala ProGln Lys Asn                 25 (SEQ ID NO:24) (SEQ ID NO:2 residues307-316) Thr Val Gln Ala Val Leu Thr Val Pro Lys                 5                  10 (SEQ ID NO:25) (SEQ ID NO:2residuea 317-327) Leu Lys Leu Ser Tyr Glu Gly Glu Val Thr                 5                  10 Lys (SEQ ID NO:26) (SEQ ID NO:2residues 360-389) Ala Gly Phe Glu Trp Asn Glu Asp Gly Ala                 5                  10 Gly Thr Thr Pro Ser Pro Gly LeuGln Pro                  15                  20 Ala His Leu Thr Phe ProLeu Asp Tyr His                  25                  30 (SEQ ID NO:27)

EXAMPLE 24 Comparison of the PEDF Peptides to Rat Serine ProteinaseSequences

PEDF-13 shows significant sequence homology with serpin proteinasemolecules. Homologous molecules include rat serine protease inhibitors 1and 2, a rat hepatocyte growth hormone-regulated protein, a rat thyroidhormone-regulated protein, and mouse contrapsin. Human, monkey, sheep,and mouse alpha-1 antitrypsin, and porcine alpha-1-antichymotrypsin,show significant but somewhat less homology. PEDF-14 shows homology withdifferent protease inhibitors, but the degree of homology is less thanthat observed for PEDF-13. These include human plasma protease C1inhibitor and human alpha-2-antiplasmin inhibitor. These results suggestthat PEDF may function as a protease inhibitor, but that it ismolecularly distinct from such inhibitors which have been describedpreviously.

PEDF shows significant homology with serpins, the family of serineprotease inhibitors that share a common reactive center near theC-terminal end, which serves as an exposed binding site that acts asbait for target serine proteinases. It is of interest that a number ofknown members of the serpin family have been shown to have neurotrophiceffects on a variety of neuronal cell types. For example, glial-derivednexin (GDN) promotes neurite outgrowth in neuroblastoma cells, as do anumber of other protease inhibitors, such as hirudin and leupeptin.Protease inhibitors also stimulate neuronal differentiation in cells ofdorsal root ganglia, sympathetic neurons, and hippocampal pyramidalneurons. It is also of interest that the production of proteinases andprotease inhibitors is stimulated by a number of known growth factors.While it remains to be determined if PEDF has protease inhibitoractivity, it is likely, since inhibitory activity has been shown to benecessary for the neurite-promoting activity of glial derived nexin. Ithas been suggested that the formation of a stable protease-GDN complex,and a consequent conformational change in GDN and/or the associatedprotease, is necessary for the neurite-promoting activity of GDN. PEDFmay well function similarly, as there are known proteinases present inthe interphotoreceptor matrix and in the developing neural retina.

EXAMPLE 25 Cloning of the PEDF cDNA

The following oligonucleotides were constructed:

5′-AGYAAYTTYT AYGAYCTSTA-3′ (SEQ ID NO:28) determined in Example 22;and:

5′-CTYTCYTCRT CSAGRTARAA-3′ (SEQ ID NO:29) determined in Example 23.

The oligonucleotides were prepared on an ABI 392 DNA/RNA Synthesizer andused as primers in a polymerase chain reaction (PCR). A human fetal eyeCharon BS cDNA library, donated by Dr. A. Swaroop, was amplified once bythe method described by Sambrook et al., 1989 In: Molecular Cloning: ALaboratory Manual 2nd ed. Cold Spring Harbor Press, Cold Spring Harbor,N.Y. and screened by PCR as described by Friedman et al. (Screening ofλgt 11 libraries In: PCR Protocols: A Guide to Methods and ApplicationsInnis, Gelfand, Sninsky and White, eds., Academic Press, pp. 253-260,1990) using a Techne thermal cycler and standard reagents (GeneAMP,Perkin-Elmer/Cetus), except that MgSO₄ was used at 3 mM.

The recovered fragment was isolated on a 3% w/v NuSieve 3:1 gel (FMCBiochemicals, Rockland Me.) using NA-45 DEAE-cellulose paper (Schleicherand Schull) and labelled with ³²P-dCTP (Amersham Corp., ArlingtonHeights, Ill.) by random priming (Random Priming kit,Boehringer-Mannheim, Indianapolis, Ind.; Prime-It Random Primer LabelingKit, Stratagene).

This probe was used to screen 200,000 pfu's of the same library.Positive clones were isolated as described by Sambrook et al., 1989 In:Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring Harbor Press,Cold Spring Harbor, N.Y., and the DNA was purified with Qiagen Maxipreparation protocols (Qiagen).

The inserts were cut out with NotI (BRL, Gaithersburg, Md.) circularizedwith T4 DNA ligase (New England Biolabs, Beverly, Mass.), transformedinto competent E. coli Epicurian Sure cells (Stratagene), and plated outon 12.5 μg/ml ampicillin/40 μg/ml X-gal agar plates.

White colonies were selected and mini-prepped (Qiagen plasmid mini-prepprotocol). Purified plasmids were digested with EcoR1/HindIII (BRL).These restriction sites were added during library construction throughthe ligation of linkers to the 5′ and 3′ ends of the insert, thusEcoRI-HindIII digestion excises the insert present in isolated plasmids.These fragments were electrophoresed on a 0.7% w/v agarose gel todetermine insert size. The plasmid possessing the largest insert, namelyπFS17, was selected for mapping and subsequent sequencing using theSequenase 2.0 sequencing kit (United States Biochemical Corp.,Cleveland, Ohio) to confirm the identity of the clone. Sequence analysiswas performed using the MacVector software package (InternationalBiotechnologies, Inc.) and the GenBank® Sequence Data Bank(Intelligenetics, Mountain View, Calif.).

EXAMPLE 26 cDNA Sequence Analysis of Human PEDF

One of the identified clones which contained an insert corresponding tothe PEDF gene was selected for mapping and subsequent sequencing using aUSB Sequenase 2.0 protocol and reagents.

Sequence analysis of πFS17 revealed a base sequence comprising SEQ IDNO:1, with a long, open reading frame (ORF) encoding the 418 amino acidsof SEQ ID NO:2, a typical ATG start codon, and a polyadenylation signal(not shown in SEQ ID NO:1). The coding sequence of the clone alignsexactly with all previously determined PEDF peptide sequences. Thededuced amino acid sequence also contains a stretch of hydrophobic aminoacids that could serve as a signal peptide. A comparison of the codingsequence and peptide sequence with the GenBank® Data Bank indicates thatPEDF is a unique protein having significant homology to the serpin(serine antiprotease) gene family, which includes human[α]-1-antitrypsin. Although some of the members of this gene familyexhibit neurotrophic activity (Monard et al., Prog. Brain Res. 58359-364, 1983); Monard, TINS 11 541-544, 1988), PEDF lacks homology tothe proposed consensus sequence for the serpin reactive domain.

EXAMPLE 27 cDNA Sequence Analysis of Mouse PEDF

An isolated cDNA incorporating the mouse cDNA was sequenced. Thesequence covering the first 376 amino acids is presented in SEQ ID NO:7.

EXAMPLE 28 cDNA Sequence Analysis of Bovine PEDF

An isolated cDNA incorporating the bovine cDNA was sequenced. Thesequence is presented in SEQ ID NO:8.

EXAMPLE 29 Construction of an Expression Vector for the Production ofRecombinant PEDF-BH

An expression vector was constructed using the plasmid πFS17, whichcontains the full-length cDNA for human PEDF as described in Example 25.The PEDF coding sequence was placed under the control of a bacteriophageλP_(L) promoter present in the plasmid pEV-vrf2 (Crowl et al., Gene 3831-38, 1985) to obtain the vector pEV-BH. This was accomplished byobtaining a BamHI-HindIII fragment of πFS17 comprising a portion of thePEDF coding region (namely, nucleotides 245 to 1490 of SEQ ID NO:1),digesting plasmid pEV-vrf2 with EcoRI-HindIII, rendering both fragmentsblunt by means of a fill-in reaction at the BamHI and EcoRI ends withDNA polymerase I (Klenow fragment), and ligating the resultantblunt-ended/compatible-ended fragments to each other. The resultantvector pEV-BH places a distance of 8 nucleotides between theShine-Dalgarno (SD) sequence and the PEDF coding region. The constructspecifies Met-Asn-Arg-IIe-Asp44---Pro418 (SEQ ID NO:3) such that aprotein of 379 amino acids, known as PEDF-BH, is encoded as indicated inSEQ ID NO:3. The amino acids at the amino terminus of the PEDF-BHprotein do not occur in native PEDF and result from the fusion ofnucleic acids during the construction of pEV-BH.

To verify production of the recombinant PEDF-BH protein by PEV-BH, theplasmid was propagated in E. coli strain RRI (Maniatis et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1982), bearing the low copy-number compatible plasmidpRK248cIts that contains a gene for encoding a temperature-sensitiveλcIAt2 repressor (Bernard et al., Methods in Enzymology 68 482-492,1979). Protein induction was performed as described in Becerra et al.,Biochem. 30 11707-11719 (1991), with the following modifications.Bacterial cells containing pEV-BH were grown in LB medium containing 50μg/ml ampicillin at 32° C. to early logarithmic phase, such thatOD_(600nm)=0.2. The temperature of the culture was rapidly increased to42° C. by incubating the flask in a 65° C. water bath, and the bacteriawere subsequently grown at 42° C. for 2-3 hours in an air-flow incubatorat 340 rpm. Aliquots were taken for absorbance readings at 600 nm.

Nascent proteins, synthesized following protein induction, wereradiolabeled. After the temperature of the culture had reached 42° C.,150 μCi of L-[³⁵S]methionine (1040 Ci/mmol, Amersham Corp., ArlingtonHeights, Ill.) were added per ml of culture, and incubation wascontinued at 42° C. for 10 minutes and 30 minutes. Cells were harvestedby centrifugation and washed with TEN buffer (10 mM Tris-HCl, pH 7.5, 1mM EDTA, and 100 mM NaCl). ³⁵S-labeled peptides from total bacterialextracts were resolved and analyzed on SDS-12% w/v PAGE followed byfluorography. A band corresponding to a 42,820 M_(T) polypeptide wasdetected 10 and 30 minutes post-induction. The size obtained for therecombinant protein expressed by pEV-BH matched the expected size forthe coding sequence subcloned in pEV-BH.

EXAMPLE 30 Construction of Expression Vectors Containing the Full-LengthPEDF cDNA

In a manner similar to that described in Example 27 for the constructionof pEV-BH, the PEDF ORF of plasmid πFS17 was placed under the control ofthe bacteriophage λPL promoter present in the plasmids pRC23 andpEV-vrf1 (Crowl et al., Gene 38 31-38, 1985). This was accomplished byobtaining the SfaNI-HindIII fragment of πFS17 comprising a portion ofthe PEDF cDNA (namely, nucleotides 107 to 1490 of SEQ ID NO:1),digesting the plasmids with EcoRI-HindIII, rendering the fragments bluntby means of a fill-in reaction at the SfaNI and EcoRI ends with DNApolymerase I (Klenow fragment), and ligating the resultantblunt-ended/compatible-ended fragments to each other. The resultingvectors pRC-SH and pEV-SH place a distance of 14 and 8 nucleotides,respectively, between the SD sequence and the PEDF coding region. Theconstruct pRC-SH encompasses the full-length PEDF ORF, and specifies aPEDF protein of 418 amino acids, with its naturally occurring aminoterminus, as set forth in SEQ ID NO:2. The construct pEV-SH encompassesthe full-length PEDF ORF, and specifies a PEDF amino-terminal fusionprotein of 425 amino acids, with Met-Asn-Glu-Leu-Gly-Pro-Arg precedingthe PEDF sequence of SEQ ID NO:2. These additional amino acids at theamino terminus do not occur in native PEDF, and the codons in pEV-SHspecifying these additional amino acids result from the fusion ofnucleic acids during the construction of pEV-SH. To verify production ofthe recombinant proteins specified by the two vectors, the vectors wereintroduced into E. coli strain RRI [pRK248cIts], and protein inductionwas performed and monitored by metabolic labeling with ³⁵S-methionineduring induction in a manner similar to that set forth in Example 27.The induced expression of the proteins specified by pRC-SH and PEV-SHhad a negative effect on bacterial cell growth. In comparison withbacterial cultures containing the parental plasmids, cultures containingpRC-SH and pEV-SH grew and divided more slowly. This negative effect onbacterial growth correlated with the distance between the initiationcodon and the SD, which may suggest that a shorter such distance resultsin more efficient translation of the recombinant protein. A 46,000 M_(T)candidate polypeptide for PEDF was not detected in the media or celllysates of bacterial cultures containing pRC-SH and PEV-SH. However, a35,000 M_(T) protein was observed in extracts of cultures containingpRC-SH and pEV-SH, but not in extracts of cultures containing parentalplasmids. This may indicate that the amino-terminal end of PEDF isprotease-sensitive and that recombinant full-length PEDF is metabolizedin this particular host. Alternatively, failure to observe theanticipated-sized recombinant PEDF proteins may reflect an experimentalartifact which could be overcome through the use of alternativeexpression vectors, hosts, inducible promoters, subcloning sites,methods of recombinant protein isolation or detection, or means ofprotein induction.

EXAMPLE 31 Method for Producing Large Quantities of RecombinantlyProduced PEDF-BH

A total of 1 g of E. coli cells containing PEDF-BH was resuspended in 50ml 20 mM Tris-HCl, pH 7.5, 20% w/v sucrose, and 1 mM EDTA. The cellswere maintained on ice for 10 minutes, sedimented by centrifugation at4,000×g, and were resuspended in 50 ml of ice-cold water for 10 minutes.Lysed outer cell walls were separated from spheroplasts bycentrifugation at 8,000×g.

The pelleted spheroplasts were resuspended in 10 ml of phosphatebuffered saline (PBS) containing 5 mM EDTA, 1 μg/ml pepstatin and 20μg/ml aprotinin. The suspension was probe-sonicated with a sonicator(Ultrasonics, Inc., model W-225) to lyse the cell membranes. Threebursts at 30 second pulses with a 30 second pause were performed whilethe sample was immersed in an ice-water bath. RNase TI (1,300 units,BRL) and DNase I (500 μg, BRL) were added to the sonicated cellsuspension, and the suspension was incubated at room temperature for 10minutes. This suspension was diluted by the addition of 40 ml ofphosphate buffered saline (PBS) containing 5 mM EDTA, 1 μg/ml pepstatinand 20 μg/ml aprotinin, and the crude inclusion bodies were sedimentedby centrifugation at 13,000×g for 30 minutes. The particulate materialconsisting of inclusion bodies was resuspended in 40 ml of PBScontaining 25% w/v sucrose, 5 mM EDTA, and 1% v/v Triton X-100,incubated on ice for 10 minutes, and centrifuged at 24,000×g for 10minutes. The washing step was repeated three times. Finally, theinclusion bodies were resuspended in 10 ml of denaturation buffercontaining 50 mM Tris-HCl, pH 8.0, 5 M guanidine-HCl, and 5 mM EDTA. Thesuspension was probe-sonicated briefly for 5 seconds in an ice-waterbath. The resulting suspension was incubated on ice for an additionalhour. After centrifugation at 12,000×g for 30 minutes, the supernatantwas added to 100 ml of renaturation buffer containing 50 mM Tris-HCl, pH8.0, 20% v/v glycerol, 1 mM DTT, 1 μg/ml pepstatin, and 20 μg/mlaprotinin, and stirred gently at 4° C. overnight to renature theprotein. The soluble and insoluble fractions were separated bycentrifugation at 13,500×g for 30 minutes.

The soluble fraction was further purified by concentrating it to 1 mlusing a Centricon 30 microconcentrator (Amicon Div., W. R. Grace & Co.,Beverly, Mass.), and dialyzing it against Buffer A (50 mM sodiumphosphate, 1 mM DTT, 20% v/v glycerol, 1 mM EDTA, 1 μg/ml pepstatin, and1 mM benzamidine) at 4° C. for 3 hours. The dialyzed extract wascentrifuged at 14,000 rpm in an Eppendorf Centrifuge (Model 5415C) forten minutes. The supernatant fraction was layered on a S-Sepharosefast-flow (Pharmacia, New Market, N.J.) medium packed into a column witha 1 ml bed volume and pre-equilibrated with buffer A. The medium waswashed with two column-volumes of buffer A. Finally, recombinant PEDF-BHwas eluted with a step gradient of 50, 100, 150, 200, 300, 400, 500, and1,000 mM NaCl in buffer A. Fractions of 1 ml were collected by gravityflow, and were dialyzed against buffer A. The 300 mM NaCl fraction,containing recombinant PEDF-BH, was stored at −20° C. The recovery infraction 300 was 50 μg per gram of packed cells, which represents 25% ofthe total protein.

Most of the PEDF-BH was recovered from the insoluble fraction bydissolving the fraction in 10 ml of 6M guanidinium-HCl in buffer B (50mM Tris-HCl, pH 8.0, 1 mM DTT, 2 mM EDTA). The solution was centrifugedat 10,000×g for 5 minutes. The supernatant was layered onto aSUPEROSE-12 (Pharmacia, New Market, N.J.) medium packed into a columnattached in tandem to a second SUPEROSE-12 medium containing column(each column 2.6 cm×95 cm) pre-equilibrated with buffer containing 4 Mguanidinium-HCl in buffer B. The flow rate was 3 ml/minute. RecombinantPEDF-BH containing fractions from the SUPEROSE-12 column were pooled anddialyzed against buffer C (4 M urea, 50 mM sodium phosphate, pH 6.5, 1mM benzamidine, 1 μg/ml pepstatin, 4 mM EDTA). The dialyzed fraction waspassed through a 0.22 μm filter (Miller-GV, Millipore Corp., Bedford,Mass.). The filtered solution was layered onto a MONO-S (Pharmacia, NewMarket, N.J.) medium packed into a column (1 cm×10 cm, d×h) andpre-equilibrated with buffer C. The medium was washed with buffer C, andrecombinant PEDF-BH was eluted with a gradient of 0-500 mM NaCl inbuffer C at 0.5 ml/minutes. Two-ml fractions were collected, and thepeak fractions of recombinant PEDF-BH were pooled. The recovery in thepooled fractions was 0.5 mg of recombinant PEDF-BH per gram of packedcells.

EXAMPLE 32

Use of Purified Recombinant PEDF-BH as a Differentiation Agent

Y79 cells (ATCC, HTB18) were grown in Eagle's Minimal Essential Mediumwith Earl's salts (MEM) supplemented with 15W v/v fetal bovine serum andantibiotics (10,000 u/ml penicillin and 10 mg/ml streptomycin) at 37° C.in a humidified incubator under 5% v/v CO₂. Cells were propagated fortwo passages after receipt from the ATCC, and then frozen in the samemedium containing 10% v/v DMSO. A few of the frozen aliquots were usedfor each differentiation experiment. All experiments were performed induplicate.

After thawing, the cells were kept, without further passaging, in theserum-containing medium until the appropriate number of cells wereavailable. Cells were collected by centrifugation and washed twofold inPBS, resuspended in PBS, and counted. At that point, 2.5×10⁵ cells wereplated into each well of a 6-well plate (Nunc, Inc., Roskilde, Denmark)with 2 ml of serum-free medium (MEM, supplemented with 1 mM sodiumpyruvate, 10 mM HEPES, 1× non-essential amino acids, 1 mM L-glutamine,0.1% v/v ITS mix (5 μg/ml insulin, 5 μg/ml transferrin, 5 ng/mlselenium, Collaborative Research, Bedford, Mass.), and antibiotics asdescribed above.

Differentiation effectors and control buffers were added 12-16 hoursafter plating, and the cultures were incubated and left undisturbed for7 days. On the eighth day, cells were transferred topoly-D-lysine-coated six-well plates (Collaborative Research, Bedford,Mass.), and the old medium was replaced with 2 ml of fresh serum-freemedium, upon attachment of the cells to the substrate. The cultures weremaintained under these conditions for up to 11 days. Post-attachmentcultures were examined daily for morphological evidence ofdifferentiation as well as quantification of neurite outgrowth using anOlympus CK2 phase-contrast microscope.

In comparison with untreated cells, only Y79 cultures that were exposedto recombinant PEDF-BH showed any significant evidence of neuronaldifferentiation. Some neurite outgrowth (below 10%) was detectable incontrol cultures treated with the same buffer used to solubilizePEDF-BH, and no evidence of differentiation was found in culturesprocessed in the same manner without the addition of PEDF-BH or buffer.Phase contrast microscopy of PEDF-BH treated cultures showed thatbetween 50-65% of the cell aggregates had neurite extensions by day 3post-attachment on poly-D-lysine. These 3-day neurite extensionsappeared as short projections from pear-shaped cells at the edges of thecell aggregates. The number of differentiating aggregates, the number ofdifferentiating cells per aggregate, and the length of the neurite-likeprocesses increased with post-attachment time. By day 5 post-attachment,about 75-85% of the aggregates showed signs of differentiation withneurites extending from most of their peripheral cells. PEDF-BH-treatedcultures reached the maximum extent of differentiation on day 7post-attachment, when 85-95% of the cells aggregate. At that time, twotypes of neuronal processes were observed, i.e., single neurites 2-3fold longer than those observed on day 3 extending from peripheral cellsof isolated aggregates, and much longer and thinner processes forming abranching network between neighbor cell aggregates. Upon extendedincubation, i.e., beyond 10 days post-attachment, there was a markeddecrease in the proportion of the network connections, and no furthergrowth of the single neurites, although the viability of the cellaggregates was not severely affected, and remained at about 75-80% indifferent experiments.

The PEDF and PEDF-BH cDNA clones not only provide means to produce largequantities of the PEDF and PEDF-BH proteins but also serve as sourcesfor probes that can be used to study the expression and regulation ofthe PEDF gene. In addition, these sequences can be used in the antisensetechnique of translation arrest to inhibit the translation of endogenousPEDF.

The recombinantly produced PEDF and PEDF-BH proteins and equivalentproteins can be used as potent neurotrophic agents in vitro and in vivo.Additional biochemical activities of these proteins as neurotrophicagents can be determined through standard in vitro tests, which willenable the development of other therapeutic uses for these proteins inthe treatment of inflammatory, vascular, degenerative and dystrophicdiseases of the retina. Given that these proteins are such potentneurotrophic agents, it can be envisioned that these proteins could bemodified for therapeutic utility in the treatment of tissues other thanthe retina, which also respond to neurotrophic factors. These proteinsmay even find more generic utility as “differentiation” factors fornon-neural tissues and certain types of cancer.

EXAMPLE 33 Treatment of Retinal Tumors

PEDF is administered, alone or in conjunction with clinical (such assurgical) procedures. PEDF is administered by intravitreal or subretinalinjection. The PEDF may be administered alone or in conjunction withcompounds such as poly(lactic acid) which facilitate a slow, “timerelease” of the PEDF. Typically, 0.01 to 10 μg of PEDF at aconcentration of 1 to 100 μg/ml in a physiologic saline solution orother buffered solution is administered per dose, and 0 to 10 doses areadministered per day. When time-release compounds are included in thecomposition, they are used at a concentration of 1 to 100 μg/ml.Treatment is continued until tumor progression is halted or reversed.

Treatment with PEDF is effective for retinal tumors such asretinoblastoma, other neuronal tumors such as neuroblastoma, or tumorsof non-neuronal origin. The treatment results in a cessation orreduction in the rate of cell division and a concomitant reduction inthe rate of tumor growth, which in turn results in tumor regression.

EXAMPLE 34 Neurotrophic Treatment of Ocular Disease

PEDF is administered, alone or in conjunction with clinical (such assurgical) procedures. PEDF is administered by intravitreal or subretinalinjection. The PEDF may be administered alone or in conjunction withcompounds such as poly(lactic acid) which facilitate a slow, “timerelease” of the PEDF. Typically, 0.01 to 10 μg of PEDF at aconcentration of 1 to 100 μg/ml in a physiologic saline solution orother buffered solution is administered per dose, and 0 to 10 doses areadministered per day. When time-release compounds are included in thecomposition, they are used at a concentration of 1 to 100 μg/ml.Treatment is continued until the pathology is halted or reversed.

Treatment with PEDF is directed at diseases of the neural retina,retinal pigmented epithelium, and other ocular tissue. Treatment resultsin enhanced survival and well-being of the photoreceptors and otherocular cells, prolonging their functional life span and delaying theonset of impaired vision and ultimate blindness.

EXAMPLE 35 Neurotrophic Treatment of Injured Nerves

PEDF is administered, alone or in conjunction with clinical (such assurgical) procedures. PEDF is administered by intravitreal or subretinalinjection. The PEDF may be administered alone or in conjunction withcompounds such as poly(lactic acid) which facilitate a slow, “timerelease” of the PEDF. Typically, 0.01 to 10 μg of PEDF at aconcentration of 1 to 100 μg/ml in a physiologic saline solution orother buffered solution is administered per dose, and 0 to 10 doses areadministered per day. When time-release compounds are included in thecomposition, they are used at a concentration of 1 to 100 μg/ml.Treatment is continued until nerve regeneration is completed.

Treatment with PEDF is directed at injuries to nerves. Treatment resultsin promotion of neurite outgrowth and nerve regeneration.

EXAMPLE 36 Transfection of Bacterial Cells

Competent E. coli Sure cells are mixed with the ligation mixture andincubated on ice for 60 minutes. The mixture is then incubated at 42° C.for 2 minutes, then 800 μl of 2% w/v BACTO Tryptone, 2% w/v BACTO YeastExtract (both supplied by DIFCO LABORATORIES), 10 mM NaCl, 10 mM MgSO₄,20 mM glucose are added and the mixture incubated at 37° C. for 60minutes. Fifty to 500 μl of the DNA mixtures are then spread onselection plates containing 12.5 μg/ml ampicillin.

The recombinant colonies are screened by miniprepping and digestion ofthe isolated DNA. Positive colonies containing the appropriate insertsare then prepared by growing the appropriate colony in 200 ml of LuriaBroth plus 12.5 μg/ml ampicillin with shaking at 37° C. overnight. Afterthe overnight incubation, the cultures are transferred to centrifugebottles and centrifuged at 2500 rpm for 10 minutes. The supernatant isremoved and the cells are resuspended in 30 ml of STET buffer (0.23 Msucrose, 5% v/v Triton-X-100, 20 mM EDTA, 50 mM Tris-HCl, pH 8) at roomtemperature. Five μl of 10 mg/ml lysozyme is added and the mixture isswirled and incubated for 5 minutes at room temperature. Each flask isthen gently swirled directly over a flame until the cells begin tocoagulate and turn white. The mixture is then transferred to aboiling-water bath for about 45 seconds. The solution is then cooled inan ice-water bath for 2 minutes, and the mixture is transferred tocentrifuge tubes and centrifuged for 15 minutes at 16,000 rpm.

The supernatant is then transferred to a clean container. Two volumes of100% ethanol are added, mixed, and the DNA precipitated at −70° C. for20 minutes. The precipitate is collected by centrifugation at 2,500 gfor 15 minutes. The ethanol supernatant is removed, and the pellet iswashed with 10 ml of cold (−20° C.) 90% v/v ethanol. Two-and-one-half mlof extraction buffer (0.2 M Tris, pH 7.5, 0.08 M EDTA, and 0.2 M KCl) isadded to the pellet, and it is resuspended, at 4° C. 100 μl of 10 mg/mlRNAse, dissolved in 0.1× TE (1 mM Tris-HCl, 0.1 mM EDTA, pH 7.6) andpretreated by boiling for 10 minutes.

EXAMPLE 37 Insect Cell Cultures

Sf9 cells are seeded into spinner flasks, containing Grace's Antheraeamedium supplied by GIBCO of Grand Island, N.Y., to an initial density of1×10⁶ cells/ml and incubated at 27° C. with constant stirring at 50 rpm.The cells are subcultured when they reach a density of 2.5×10⁶ cells/ml,approximately 2 to 3 times a week, and are diluted 1 in 5.

EXAMPLE 38 Cloning Genes into pAC373

Two μl of pAC373 are combined with 25 μl of 10× BamHI restriction enzymebuffer, 20 units of BamHI and sterile distilled water to bring thevolume to 250 μl, and incubated at 37° for at least 3 hours. After theplasmid is digested. It is dephosphorylated by adding one unit of calfintestinal alkaline phosphatase (CAP)/μg of DNA and incubated for 30minutes at 37° C. The CAP is then inactivated by adding EDTA to 25 mMand SDS to 0.5% w/v and incubated at 65° C. for 15 minutes. The solutionis then extracted with an equal volume of phenol/chloroform/ isoamylalcohol (25:24:1).

The aqueous phase is collected, and 125 μl of 7.5 M ammonium acetate and800 μl of 95% v/v ethanol are added and mixed. The DNA is precipitatedat −70° C. for 10 minutes. The precipitate is then collected bycentrifugation by centrifugation at 12,000 rpm for 10 minutes. Thepellet is rinsed with (−20° C.) 90% v/v ethanol. The ethanol is thenremoved and the DNA is resuspended in 50 μl of 10 mM Tris-HCl, 1 mMEDTA, pH 7.6 (1× TE).

EXAMPLE 39 Ligation of PEDF to pAC373

A purified DNA fragment containing the PEDF gene is ligated to thetransfer vector. Two-hundred ng of digested pAC373 are mixed with anequal molar concentration of PEDF containing fragment. Ligation buffer(50 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 10 mM dithiothreitol, 0.5 nMspermidine, 2 mM ATP, 2.5 mM hexamine cobalt chloride and 20 μg/ml BSA)and 10 units of T4 DNA ligase is added. Water is added to a total volumeof 10 μl. The mixture is incubated at 14° C. for 3 hours.

EXAMPLE 40 Transferring Genes into the AcMNPV Genome

Sf9 cells are seeded into 25 cm flasks at a density of 2.0×10⁶cells/flask in Grace's Antheraea medium. The cells are allowed to attachfor at least one hour. One μg of MNPV DNA is added to 2 μg of plasmidDNA, which contains the PEDF gene. The medium is removed from the flaskand replaced with 0.75 ml of Grace's medium plus 10% v/v fetal bovineserum and antibiotics (50 μg/ml gentamicin sulfate, 2.5 μg/mlamphotericin). 7.5 ml of transfection buffer (25 mM HEPES, pH 7.1, 140mM NaCl, 125 mM CaCl₂) is added to the DNA solution and mixed. The DNAsolution is added to the Grace's medium, already in the cell cultureflasks.

Calcium phosphate precipitates form due to the calcium chloride in thetransfection buffer and the phosphate in the medium. The flasks areincubated at 27° C. for 4 hours, after which time the medium is removedfrom the flasks, and the cells are rinsed with fresh TNM-FH (Grace'smedium plus 3.3 g/l YEASTOLATE and 3.3 g/l Lactalbumin hydrolysate, bothfrom DIFCO LABORATORIES) plus 10% v/v fetal bovine serum andantibiotics, as described previously, and 5 ml of TNM-FH plus 10% v/vfetal bovine and antibiotics, as described previously, is added to thecells. The cells are incubated for 4-6 days. When the infection is at anadvanced stage, the virus is plated on fresh monolayers, and therecombinant viruses are plaque-purified.

EXAMPLE 41 Identification of Recombinant Proteins

Plaque-purified virus is screened by radiolabelling. The recombinantproteins are identified on SDS-PAGE gels. Sf9 cells are seeded at 6×10⁵cells/well in a 24-well culture plate. The cells are allowed to attachfor an hour. The medium is then removed and overlaid with mediumcontaining the viral stock and incubated for 1 hour at 27° C., afterwhich time the viral inoculum is removed and replaced with 500 μl ofcomplete medium. The cells are incubated for 48 hours at 27° C. At theend of this incubation, the medium is removed. 200 μl of methionine-freeGrace's medium is added to the cells, and the cells are incubated for 60minutes, after which time the medium is replaced with 200 μl of freshmethionine-free Grace's medium to which 10 μCi of ³⁵S-methionine isadded. Cells are incubated at 27° C. for 6 hours and then harvested bycentrifugation. The supernatant is collected, and the cells areresuspended in 62.5 mM Tris, pH 6.8, 2% w/v SDS, 10% v/v glycerol,0.001% w/v bromophenol blue, and 0.1 M 2-mercaptoethanol. An equalvolume of 126 mM Tris, pH 6.8, 4% w/v SDS, 20% v/v glycerol, 0.002% w/vbromophenol blue, and 0.2 M 2-mercaptoethanol is added to thesupernatant. Samples are then boiled for 3 minutes and then they aresubjected to electrophoresis and autoradiography of the gel to identifyproteins secreted into the medium and proteins that are not secreted.Controls of uninfected cells and cells infected with wild-type virus areincluded.

EXAMPLE 42 Chromosomal Localization of the PEDF Gene: SouthernHybridization

Human-hamster somatic cell hybrid DNAs were used to identify thechromosomal location of the PEDF gene. Twenty-five hybrids werecharacterized by karyotype analysis by the method described by Carlocket al. (Somatic Cell Mol. Genet. 12 163-174, 1986), and the chromosomecontent of current passages of each cell line was determined by Giemsabanding analysis of 20 metaphases. DNA samples were subjected torestriction fragment length analysis using a number of restrictionenzymes to establish a polymorphism within the PEDF gene. RsaI was foundto be suitable for such use. DNA samples were digested with RsaI, and 8μg of each digested DNA sample was fractionated by electrophoresis in a1% w/v agarose gel and transferred onto neutral nylon membranes (BIODYNEA sold by Pall Corp.).

Two 20-mer PCR primers were synthesized from the middle of thetranslated region of the PEDF cDNA sequence. The primer pair:

5′-CTGGGAGCGG ACGAGCGAAC-3′ (SEQ ID NO:30) located at SEQ ID NO:1396-415 (primer 353) and

5′-TGGGGACAGT GAGGACCGCC-3′ (SEQ ID NO:16) located on the antisensestrand of SEQ ID NO:1 at 1043-1062 (primer 354), amplifies a 667 bpproduct from the PEDF cDNA. PCR amplification was carried out for 30cycles (each cycle: 94° C., 1 minute; 55° C., 1 minute; and 72° C., 1minute) by methods well known to those skilled in the art. Fiftynanograms of the amplified product was labeled by random priming using arandom PRIME-IT kit purchased from Stratagene. Unincorporatednucleotides were removed using Stratagene's Cloning Systems NUCTRAP PUSHcolumns. The nylon filters were prehybridized for 30 minute at 65° C. inQUIKHYB solution (Stratagene) followed by hybridization for 1 hour at65° C. in the same solution supplemented with 1×10⁶ cpm/ml radiolabeledPEDF probe, by methods well known to those skilled in the art.Posthybridization washes were performed for 30 minutes at 25° C. using300 mM NaCl, 35 mM sodium citrate, pH 7.0, 0.1% w/v SDS (2×SSC, 0.1%SDS) and 30 minute at 65° C. in 2×SSC/0.1% SDS. Filters were exposed toHyperfilm-MP (Amersham, Ill.) with an intensifying screen.

Somatic cell hybrid DNAs were scored on Southern blots for the presenceor absence of human-specific hybridization with the PEDF 667 bp PCRfragment. The PEDF cDNA probe identifies 6.5, 6.0, 5.0, and 4.8 kb humanrestriction fragments, as can be seen in the human genomic DNA controllanes in each blot. Of the 25 somatic cell hybrids examined, only 2contained a PEDF-specific band of an approximate 5.0 kb molecular size.These hybrids are the only ones which contain human chromosome 17. Apositive correlation therefore is observed between the presence of thisband and human chromosome 17.

EXAMPLE 43 Chromosomal Localization of the PEDF Gene: PCR Assay

DNAs from human-rodent somatic cell hybrid panels (1 and 2) werepurchased from the NIGMS Human Genetic Mutant Cell Repository at theCoriell Institute for Medical Research (Camden, N.J.). PCR andchromosomal localization studies (Sambrook et al., 1989 In: MolecularCloning: A Laboratory Manual 2nd ed. Cold Spring Harbor Press, ColdSpring Harbor, N.Y.) were performed using primers 3 and 4 and hybridDNAs from NIGMS panel 1. Primers 3 and 4 were designed from the 3′translated region of the PEDF cDNA using OLIGO primer analysis software(National Biosciences, Plymouth, Minn.). Primer 1 sequence:

5′-CACCTTAACC AGCCTTTATT C-3′ (SEQ ID NO:31) is located in SEQ ID NO:1at 1281-1304 and primer 2 sequence:

5′-AACCTTACAG GGGCAGCCTT CG-3′ (SEQ ID NO:32) and is located on theantisense strand in SEQ ID NO:1 at 1438-1459, amplified a 179-bp productfrom the PEDF cDNA and human genomic DNA. The PCR reaction contained 10mM Tris-HCl, pH 8.3, 50 mM KCl, 80 μM of each dNTP, 1 mM MgCl2, 200 ngof each primer, 100 ng of genomic DNA, and 1.25 units of AMPLITAQ(Perkin Elmer Cetus). Amplification was carried out for 40 cycles (eachcycle consist-ing of 94° C., 1 minute; 57° C., 1 minute; and 72° C. for2 minute).

For panel 2, two additional 24-mer PCR primers from the 3′-untranslatedregion of the PEDF cDNA were designed. Partial purification anddesalting of this primer pair were accomplished by passing theoligonucleotide through SEPHADEX G-25 (Pharmacia, Upsala Sweden) packedinto a column. Primer 498:

5′-TATCCCAGTT TAATATTCCA ATAC-3′ (SEQ ID NO:33) is located at SEQ IDNO:1 at 1374-1397 and primer 499:

5′-TTGTATGCAT TGAAACCTTA CAGG-3′ (SEQ ID NO:34) is located on theantisense strand of SEQ ID NO:1 at 1448-1472 defines a 99-bp domain inthe 3′ noncod-ing region of the human PEDF cDNA sequence. PCR wasperformed in a 100-μl reaction containing 10 mM Tris-HCl, pH 8.3, 50 mMKCl, 100 μM of each dNTP, 1.5 mM MgCl2, 200 ng of genomic DNA from eachhybrid, 200 ng of PEDF primers 5 and 6, and 1.25 units of AMPLITAQ(Perkin Elmer Cetus). DNA was subjected to preamplification heating at94° C. for 10 minute, after which amplification was carried out for 20cycles. Each cycle consisted of 1 minute denaturation at 94° C., 1minute annealing at 47° C., and 2 minute extension at 72° C. A finalextension was carried out at 72° C. for 7 minute. One microliter of theamplified product was reamplified using the above procedure.

Using primers 3 and 4, a 179 bp product was amplified from human genomicDNA which was absent from hamster and mouse genomic DNA. In ahuman-rodent somatic cell hybrid panel (NIGMS MAP 1), the 179 bp productwas observed in somatic hybrids 1-15, which contain a number of humanchromosomes, including chromosome 17, but not in hybrids 16-18, which donot contain chromosome 17. A second panel, NIGMS MAP 2, also wasinvestigated using PEDF primers 498 and 499, which yield a 99 bpproduct. Each hybrid of this panel contains only one human chromosome.Of the 24 hybrids tested, only GM/NA 10498 was positive and was the onlyone to contain human chromosome 17. Amplification was seen with humangenomic DNA but not with mouse and hamster DNAs.

EXAMPLE 44 Chromosomal Localization of the PEDF Gene: Fluorescence insitu Hybridization (FISH)

A 7-kb fragment of the PEDF gene from a cosmid clone was prepared andpurified using Quiagen plasmid protocol (Quiagen Inc., Chatsworth,Calif.) and digested with NotI (Gibco BRL, Gaithersburg, Md.), and theinsert size was determined on a 0.5% w/v agarose gel. FISH was performedat Bios Laboratories after the PEDF probe was labeled by nicktranslation with dUTP digoxigenin as described by Lichter et al.(Science 247 64-69, 1990). A chromosome 17 centromeric-specific probe,D17Z1 (Oncor, Gaithersburg, Md.), which hybridizes to a 171 bpα-satellite tandem repeat, was used as a marker in a cohybridizationstudy. The D17Z1 probe was nick translated and labeled with biotin-dATP,by methods well known in the art. The PEDF genomic DNA probe wasdetected using antidigoxigenin-fluorescein-conjugated F(ab) fragments;D17Z1 was detected with avidin-conjugated fluorescein-isothiocyanate(FITC). Chromosomes were counter-stained with propidium iodide. Eightseparate hybridizations were performed for each probe.

Fluorescence in situ hybridization analyses of eight metaphasechromosome spreads using a digoxigenin-labeled PEDF probe revealed that50% of the cells demonstrated specific fluorescent signals on chromosome17. The identity of the chromosome and its specificity were confirmedsince the PEDF probe clearly cohybridized with D17Z1, a probe specificto a-satellite tandem repeats in the centromere of chromosome 17. Ofimportance as well is that, in each case, the genomic probe gave aspecific hybridization signal at the terminal end of the short arm ofchromosome 17 (region 13.1).

EXAMPLE 45 Subchromosomal Localization of the PEDF Gene: PCR Assay

DNAs from a deletion mapping panel of eight somatic cell hybridscontaining different regions of human chromosome 17 (see Guzzetta etal., Genomics 13 551-559, 1992) were used to further sublocalize thePEDF gene. PCR conditions for panel 1 were used in this assay, includingprimers 3 and 4. Up to 600 ng of DNA for the chromosome 17 HO-11 hybridwas used in the PCR assay.

The use of human-rodent cell hybrids containing defined regions ofchromosome 17 (see Guzzetta et al., Genomics 8 279-285, 1991; Guzzettaet al., Genomics 13 551-559, 1992) allows accurate subchromosomallocalization of the PEDF gene. PCR primers 3 and 4, which yield a 179 bpproduct, again were used in this study. The PCR amplification productwas seen with four of these hybrids: MH22-6 contains the entirechromosome; DHA-4 has a deletion in the p11.2 region; 88-H5 and 357-2Dretain much of the p arm, including the 13.1 region. Two hybrids areseen to be negative: L(17n)C, which contains only 17q; and MH41, whichhas only the distal q arm. The hybrid 12.3B, however, which retains 17pand the proximal q region, is positive. Finally and of greatestsignificance, the hybrid HO-11, which is missing only the 17p13.1 to17pter region, is negative. Together, these data localize the PEDF geneto 17p13.1-pter.

Additional experiments indicate that PEDF is located on chromosome 11 inmice. The mouse chromosome 11 is syntenic with human chromosome 17.

EXAMPLE 46 Expression Analysis: Northern Hybridization

RNA was extracted from human Y79 retinoblastoma cells. The cells weregrown in suspension culture under the following three conditions: (A)medium (MEM, Mediatech) containing 15% v/v fetal bovine serum(Y79-control); (B) serum-free, defined medium (MEM, Y79-SF); (C)serum-free, defined medium containing 5% v/v bovine solubleinterphotoreceptor matrix components (Y79-IPM) as previously describedby Tombran-Tink et al., J. Comp. Neurol. 317 175-186, 1992). Under afourth condition (D), the cells were stimulated with 5% v/v bovine IPMfor 1 week in suspension culture and then attached and allowed todifferentiate for 10 days (differentiated Y79) as previously described(Tombran-Tink et al., J. Comp. Neurol. 317 175-186, 1992). Approximately95% of these cells differentiate into a neuronal phenotype under thiscondition. Cells maintained under conditions A-C are morphologicallyundifferentiated clusters that remain in suspension. Cells under allfour conditions were harvested at a total of 17 days in culture andpoly(A)⁺ RNA extracted by the method of Sambrook et al. (1989 In:Molecular Cloning: A Laboratory Manual 2nd ed. Cold Spring Harbor Press,Cold Spring Harbor, N.Y.).

Human fetal RPE cells were kindly provided by Dr. Dean Bok (Jules SteinInstitute, Univ of Cal., L.A.) These cells were obtained from 20- to21-week-old donors and were cultured in SF-defined medium for 6 monthsprior to isolation of poly(A)⁺ RNA (Sambrook et al., 1989 In: MolecularCloning: A Laboratory Manual 2nd ed. Cold Spring Harbor Press, ColdSpring Harbor, N.Y.). Two micrograms of poly(A)⁺ RNA from each samplewas electrophoresed on a 1% v/v formaldehyde denaturing gel, transferredonto neutral nylon membrane, UV cross-linked, and hybridized with thePCR-amplified 667 bp PEDF cDNA probe, by methods well known to thoseskilled in the art. Hybridization procedures were similar to those usedfor Southern hybridization of somatic cell hybrid panels.

PEDF was originally detected as a secreted protein in the medium ofhuman fetal retinal pigment epithelial (RPE) cells (Tombran-Tink et al.,Inves. Ophthalmol. Vis. Sci. 53 411-414, 1989). These cells demonstratea single 1.5-kb mRNA on Northern blots. Surprisingly, undifferentiatedY79 retinoblastoma cells demonstrate a 1.5-kb message even though littleif any PEDF protein can be detected in the medium. Cells grown insuspension culture for 17 days with 15% v/v fetal bovine serum,SF-defined medium, and 5% v/v soluble components of the bovineinterphotoreceptor matrix (IPM) all express the PEDF message atsubstantial but varying levels and all remain morphologicallyundifferentiated. In contrast, induction of a marked neuronal phenotypeand total inhibition of PEDF message are seen in Y79 cells that weretreated with soluble components of the bovine IPM for 1 week and thenmaintained in attachment culture for 10 additional days.

In addition Northern blots were performed using mRNA from a variety ofhuman adult and fetal tissues, as described above. The results indicatethat PEDF mRNA is present in retina, heart, brain, placenta, lung,liver, skeletal muscle, kidney, pancreas, thymus, prostate, testis,ovary, small intestine and colon. No significant mRNA was detected inadult kidney or spleen, however, PEDF is expressed in abundance in fetalkidney. The most abundant expression of PEDF is in liver, skeletalmuscle, testis and ovary.

In brain tissue PEDF is expressed in the amygdala, caudate nucleus,corpus callosum, hippocampus, hypothalmus, substantia nigra, subthalmicnucleus, thalamus and pineal. By Western blots, PEDF is seen in RPE-CMfrom a number of species including humans, cow, monkey and chicken.Large quantities of PEDF can also be detected in the vitreaous humor ofthese vertebrates.

EXAMPLE 47 Isolation of Genomic Clones: Screening Genomic Libraries

Bluescript plasmid containing a 1.5 kb PEDF cDNA insert was digestedwith Ecor1 and HindIII (BRL). Twenty-five ng of PEDF cDNA insert,purified by gel electrophoresis was labelled with α-³²p dCTP (Amersham)by random priming (RANDOM PRIME IT kit from Stratagene). Unincorporatednucleotides were removed by Stratagene's NUC TRAP push columns. Thelabelled probe was used to screen two genomic DNA libraries: a cosmidlibrary constructed from MboI partial digests of human placental DNA(Clonetech) and a human placental genomic library constructed in λ DASHII (Stratagene). Positively hybridizing clones were isolated by standardmethods (Troen Methods in Enzymology 151 426, 1987; Sambrook et al.,1989 In: Molecular Cloning: A Laboratory Manual 2nd ed. Cold SpringHarbor Press, Cold Spring Harbor, N.Y.) and the DNA purified with Qiagenmaxi preparation protocols (Qiagen).

Southern blot analysis of the purified clones revealed the presence oftwo strongly hybridizing fragments: a 7.1 kb BamHI fragment from thecosmid clone and a 7.2 kb Not1 fragment from the λ DASH II clone. Thesewere selected for subcloning in Bluescript and DNA sequencing.

EXAMPLE 48 Isolation of Genomic Clones: Cloning by PCR

Four sets of primers, designed from the internal coding region of thePEDF cDNA sequence were synthesized using an ABI 392 DNA/RNA synthesizerfor use as primers in polymerase chain reaction (PCR) experiments. Theprimer sequences are as follows: primer 603:

5′-ACAAGCTGGC AGCGGCTGTC-3′ (SEQ ID NO:9) is located in SEQ ID NO:1 at271-290; primer 604:

5′-CAGAGGTGCC ACAAAGCTGG-3′ (SEQ ID NO:10) is located on the antisensestrand in SEQ ID NO:1 at 570-590; primer 605:

5′-CCAGCTTTGT GGCACCTCTG-3′ (SEQ ID NO:11 ) is located in SEQ ID NO:1 at570-590; primer 606:

5′-CATCATGGGG ACCCTCACGG-3′ (SEQ ID NO:12) is located on the antisensestrand in SEQ ID NO:1 at 829-848; primer 2213:

5′-AGGATGCAGG CCCTGGTGCT-3′ (SEQ ID NO:13) is located in SEQ ID NO:1 at114-133; primer 2744:

5′-CCTCCTCCAC CAGCGCCCCT-3′ (SEQ ID NO:14) is located on the antisensestrand in SEQ ID NO:1 at 224-244; primer 2238:

5′-ATGTCGGACC CTAAGGCTGT T-3′ (SEQ ID NO:15) is located in SEQ ID NO:1at 846-866; primer 354:

5′-TGGGGACAGT GAGGACCGCC-3′ (SEQ ID NO:16) is located on the antisensestrand in SEQ ID NO:1 at 1043-1062. Standard reagents (obtained fromGeneAMP, Perkin-Elmer/Cetus of Norwalk, Conn.) and 500 ng of humangenomic DNA obtained from peripheral blood lymphocytes were used. PCRreactions were carried out at a number of different annealingtemperatures until only single amplified products were obtained. Theprimer pairs 603:604 amplified a single 2 kb PCR product (jt108);605:606 amplified a single 3.3 kb PCR product (jt109); 2213:2744amplified a single 2.3 kb PCR product (jt115) and 2238:354 amplified asingle 1.5 kb PCR product (jt116).

Seven clones isolated either from genomic libraries or by PCR-mediatedcloning were sequenced and used to characterize the exon structure ofthe PEDF gene and to define the intron/exon junction sequences. Theconventional method of cloning was replaced by PCR-mediated cloningbecause of instability and rearrangement of the gene in both cosmid andλ genomic libraries.

Two positively hybridizing clones, jt101 (7.1 kb) and jt106 (7.2 kb)were isolated from a cosmid and λ DASHII genomic libraries respectively.Four clones of jt108 (2 kb), jt109 (3.3 kb), jt115 (2.3 kb) and jt116(1.5 kb) represented PCR products of human genomic DNA. Ir117 (6 kb)clone was obtained from an exon 1-labeled positively hybridizing BamHIfragment from human genomic and P147 DNA. Two P1 clones, P1-11 and P1-47containing the entire PEDF gene were also isolated and intron/exonboundaries sequenced.

EXAMPLE 49 Isolation of Genomic Clones: Identification of P1 Clones

Two sets of primers JT10-UP01:JT10-DP01 corresponding to bases 6536-6559of jt106 genomic sequence and 1590:1591 corresponding to bases 1-89 ofSEQ ID NO:1 were used in PCR reactions to isolate P1 clones (GenomeSystems). The primer sequences are as follows: primer Jt10-UP01:

5′-GGTGTGCAAA TGTGTGCGCC TTAG-3′ (SEQ ID NO:17) is located in SEQ ID NO:4 at 6536; primer JT1-DP01:

5′-GGGAGCTGCT TTACCTGTGG ATAC-3′ (SEQ ID NO:18) is located in SEQ ID NO:4 at 7175; primer 1590:

5′-GGACGCTGGA TTAGAAGGCA GCAAA-3′ (SEQ ID NO:19) is located in SEQ IDNO:1 at 1-25; and primer 1591:

5′-CCACACCCAG CCTAGTCCC-3′ (SEQ ID NO:20) is located in SEQ ID NO:1 at71-89. Several positive clones were isolated by PCR and two, designatedP1-11 and P1-47, were subjected to Southern blotting analysis and PCRassays to confirm the presence of the entire PEDF gene and splicejunctions. Primer pairs encompassing contiguous stretches of the PEDFcDNA sequence were used to amplify products from P1-11. The primers wereas follows: primers 601 and 1591 (spanning bases 1-89 of SEQ ID NO:1;601 is the sense strand and includes bases 1-25; 1591 is the antisensestrand and includes bases 69-89), primer 2213 (SEQ ID NO:1 at 114-133)and 2744 (antisense strand in SEQ ID NO:1 at 224-244); 603 (SEQ ID NO:1at 271-290) and 604 (antisense strand in SEQ ID NO:1 at 570-590); 605(SEQ ID NO:1 at 570-590) and 606 (antisense strand in SEQ ID NO:1 at829-848); 2238 (SEQ ID NO:1 at 846-866) and 354 (antisense strand in SEQID NO:1 at 1043-1062) and 356:499 (spanning bases 1034-1472 of SEQ IDNO:1; 356 is the sense strand and includes bases 1043-1043; 499 is theantisense strand and includes bases 1448-1472). The products obtainedwere 89 bp, 2.3 kb, 2 kb, 3.3 kb, 1.5 kb and 900 bp, respectively. Theproducts were sequenced with an automated fluorescent sequencer toconfirm splice junctions of the PEDF gene from sequences obtained fromnon-PI clones.

EXAMPLE 50 Characterization of Genomic Clones DNA Sequencing:Dideoxynucleotide Termination Method

jt101 and jt106 were purified by gel electrophoresis and subcloned intothe BamHI and NotI sites respectively, of pBluescript II SK⁺ vectors(Stratagene). These were used to transform XL-I Blue competent cells(Stratagene). Transformants were isolated and subcloned. The clones wereblunt ended using T4 DNA polymerase, gel purified and subcloned into theEcoRV site of pBluescript II SK⁻ (Stratagene) and used to transform XL-Iblue cells. Nested deletions were generated from both the T7 and T3 endsof the subclones using ExoIII and SI nuclease (Lark Sequencing Co.).Plasmid DNA was prepared using a modified alkaline lysis procedure anddeletions clones size selected for DNA sequencing by electrophoresis onagarose gels. DNA sequencing (Lark Sequencing Co.) was performed usingstandard dideoxynucleotide termination method and sequencing reactionsanalyzed on 6% w/v polyacrylamide wedge gels containing 8 M urea.

jt108, jt109, jt115 and jt116 (PCR products) were cloned into themodified EcoRV site of the PT7 Blue vector (Novagen). These weresubsequently used to transform Nova Blue cells (Novagen) such that bothorientations of the insert into the vector were obtained. Nesteddeletions were then generated from the reverse end minilysates usingExoIII and SI nuclease and sequenced as above.

The sequence analysis of all the above clones the structure and size ofexons and introns of the human PEDF gene were determined. Exon/intronjunctions were established by comparing genomic and cDNA sequence ofPEDF and by identifying consensus splicing sites (Senapathy et al.,Methods in Enzymology 183 252, 1990). The analysis indicates that thehuman PEDF gene is approximately 16 kb in length and is composed of 8exons ranging in size from 92 nt to 377 nt and 7 introns ranging from0.4 kb to 6 kb (Table 1). The 5′ splice donor and 3′ splice acceptorsites in all junctions conform to the GT/AG consensus. Exons aredistributed unevenly throughout the gene and the largest intron of 6 kblong is located between exon 1 and exon 2. No significant patterns wereseen in the spatial organization of exons, in the distribution ofintrons or in the occurrence of certain types of splice junctions toinfer a unique evolutionary relationship among any subset of exons.

EXAMPLE 51 Characterization of Genomic Clones DNA Sequencing:Fluorescent Automated DNA Sequencing

Fluorescent sequencing was performed using an ABI model 370A instrumentconnected to an Apple Macintosh ci and ABI's 373 A sequencing software.The sequencing was performed using ABI's Taq DyeDeoxy Terminator cyclesequencing kit following the manufacturer's protocol. In general, 0.5pmoles of template obtained from PCR products of the P1-10 clone and 3pmoles of primer were used per sequencing reaction. All other detailsare provided in the ABI's manual included in the sequencing kit.

jt101: Sequence analysis of this 7.1 kb BamHI fragment contained themost 3′ end of the PEDF gene. Exon 7 (SEQ ID NO:1 at 903-1113) and exon8 (SEQ ID NO:1 at 1114-1503) of 211 bp and 377 bp, respectively, werecontained in this clone. Intron 6 and intron 7 were also sequenced fromjt101. Intron 7 was intact and found to be 444 bp in length while intron6 was found to be somewhat rearranged.

jt106: Sequence analysis of this 7.2 kb NotI fragment indicated onlysequences present in the most 5′ end of the PEDF gene. This clonecontained the promoter of the PEDF gene as well as exon 1 representing109 bp (SEQ ID NO:1 at 1-109) and an incomplete intron 1 of 535 bp. Wewere unable to obtain specific PCR amplification products for thisintron from either total human genomic DNA or the PI clones suggestingthat the size of the first intron was rather enormous.

jt108: The PCR clone jt108 contained a 2 kb PCR product amplified usingprimer 603:604 and contained most of exon 3 and exon 4. Intron 3 andintron 4 of 980 bp and 689 bp, respectively, were sequenced from thisclone.

jt109: This 3.3 kb clone representing PCR product obtained with primers605:606 contains most of exon 5 and exon 6 (bases 555-758 of SEQ IDNO:1). The 3 kb intron 5 is also present in this clone.

jt115: The 2.3 kb clone JT115 obtained from the PCR product amplifiedusing the primer pair 2213:2744 contained exon 2 and intron 2 which is2.9 kb in length.

jt116: Sequence analysis of this PCR clone obtained with primers 2238and 354 revealed an intact 1.5 kb intronic sequence representing intron6 previously determined from sequence analysis of jt101.

P1-11: The intron-exon boundaries of the PEDF gene in the P1-11 cloneusing primers designed from exon sequences flanking each intron havealso been sequenced. Approximately 200 bp on either side of thejunctions were sequenced and these align perfectly with the sequenceobtained from the above clones. All splice junctions sequences wereconfirmed as well as the sizes of introns and exons.

Part of the genomic DNA sequence is presented in SEQ ID NOs: 4 to 6.

EXAMPLE 52 Characterization of Genomic Clones DNA Sequencing: RACE

For RACE (Rapid amplification of cDNA ends) (Frohman In: PCR Protocols:A Guide to Methods and Applications pp 28-38, Innis, Gelfand, Sninskyand White eds., Academic Press, 1990) experiments 1.0 μg of total humanretina RNA was dried with 20 nanograms of primer 1590 (SEQ ID NO:1 at1-25). Reverse transcriptase, reaction buffer and dNTP solution (BRL)were added to a final volume of 20 μl. The reaction was carried out at42° C. for 30 minute followed by a 5 minute incubation at 55° C.Templates were tailed with poly (A) using terminal deoxytransferase(BRL). Sequences corresponding to the 5′ end of mRNA were then amplifiedby PCR using specific primer 1591. The product obtained from the PCRreaction was sequenced directly using an ABI automated fluorescentsequencer.

EXAMPLE 53 Gene Expression Analysis

Multiple human tissue mRNA Northern blots (Clonetech) with 2 μg poly(A)RNA per lane were hybridized with a radioactively labeled 667 bp PCRamplified PEDF product (Tombran-Tink et al., Genomics 19 266-272, 1994).The blots were prehybridized for 30 minute in Stratagene's QUIKHYBsolution at 68° C. The labeled probe was added to this solution andhybridization continued at the same temperature for 1 hour. Hybridizedblots were washed twice for 15 minute with 2×SSC/0.1% w/v SDS at roomtemperature and once for 30 minute with 0.1×SSC/0.1% w/v SDS at 68° C.The blots were exposed for 6 hours at −70° C. using XAR-5 film (Kodak)and intensifying screens.

EXAMPLE 54 Evolutionary Conservation Analysis

Eight μg of genomic DNA from lymphocytes of a variety of speciesincluding mammalian and primate species (BIOS laboratories, New HavenConn.) was digested with EcoR1 and separated in 1% w/v agarose gels. Thegels were transblotted and membranes containing the digested DNAhybridized using the same procedure as for the Northerns.

These studies indicated that the sequence of the PEDF gene is highlyconserved in avian, mammals and primates, as indicated by Northern blotswhich are performed under stringent conditions. Under weakerhybridization conditions, the PEDF gene from species, lower on theevolutionary scale also are identified, this includes Drosophila.

EXAMPLE 55 Expression and Secretion of the PEDF in RetinalInterphotoreceptor Matrix

Monkey eyes were obtained through the courtesy of the Office ofBiologics, FDA. The procedures for establishing RPE cells in culture anddescribing their differentiated characteristics have been publishedpreviously (Pfeffer, 1990. In Progress in Retinal Research. N. Osborneand G. J. Chader editors. Pergamon Press, Oxford UK. 251-291).Conditioned medium and cultured monkey RPE cells were used from the 1st,2nd, 5th, 10th and 15th passages of the cells. The cells were routinelymaintained in Minimal Essential Medium (MEM, Eagle's salts, Mediatech,Hearndon, Va.) containing 0.5% v/v fetal calf serum (FCS, GIBCO, GrandIsland, N.Y.). Cells from confluent monolayer cultures were scraped fromflasks, solubilized in phosphate-buffered saline (PBS, pH, 7.4)containing 0.5% v/v Triton-X (Sigma Chemical Co., St. Louis, Mo.) andstored at −70° C. for Western blot analysis. For Northern blot analysis,the following sources of RNA were used: 1) cultured fetal and adulthuman RPE cells (Flannery et al., Exp. Eye Res. 51 717-728, 1990), 2)human RPE cell explants from 21 week gestational donors (obtained fromDr. W. O'Day, Jules Stein Eye institute, UCLA,) 3) human and monkeyretina (Clonetech), 4) 1st and 10th passaged monkey RPE cell cultures(eyes obtained courtesy of In Vitro Vaccine Testing Section, FDA,Bethesda, Md.).

Conditioned medium (CM) from cultured fetal and adult human RPE cellswas obtained from monolayer cell cultures grown as previously described(Carlson et al., Biochemistry 31 9056-9062, 1992). CM from young adultmonkey and primary cultures of 11 day old chick embryo were kind giftsof Dr. Bruce Pfeffer (NEI) and Margaret Koh (Univ. of Maryland MedicalSchool), respectively. The medium was conditioned for 7 days byconfluent monolayers of RPE cells, filter-sterilized and stored at −70°C. until used. Medium conditioned by confluent monolayer of monkey RPEcells after the 1st, 2nd, 5th, 10th and 15th passage was used forWestern blot analysis to study the secretion of PEDF with successivecell passages.

Soluble IPM preparations were obtained from adult human (obtained fromthe Clinical Branch, NEI), fetal bovine (10 weeks gestation) and adultbovine eyes (obtained from Trueth and Sons, Baltimore, Md.) aspreviously described (Tombran-Tink et al., J. Comp. Neurol. 317 175-186,1992). Briefly, the anterior segment, lens and vitreous were removed;the resultant eye cups and retina were gently rinsed with 0.5 ml of PBSsolution. This preparation, which contained soluble IPM components wascentrifuged and the supernatant stored at −70° C. RPE cells were removedfrom the eye cup by vigorous repeated pipetting of a small amount of PBSagainst the cells in situ to detach them from the underlying Bruch'smembrane. The retina and the RPE cells were subsequently solubilized inSDS sample buffer to be used for Western blot analysis.

Protein concentrations were determined using the BCA assay (PierceChemical Co., Rockford, Ill.) according to the manufacturer'sspecifications. Samples of conditioned medium, IPM or cell extracts werelyophilized and 15 μg of each reconstituted in SDS sample buffer andboiled for 5 minutes. The proteins were fractionated on 10% w/vpolyacrylamide gels at 25 mAmps/gel constant current for 3 hours(Laemmli, Nature 227 680-685, 1970). Five μg samples of humanα-1-antitrypsin and β-actin (Sigma Chem. Co.) were included on the gels.The resultant gels were either stained with Coomassie Brilliant Blue(Sigma Chem. Co.) for visualization of the electrophoretic proteinprofile or transblotted onto nitrocellulose membranes for Western blotanalysis.

Two dimensional gel analysis of 1) purified PEDF protein from CM ofcultured fetal RPE cells as previously described (Tombran-Tink et al.,Exp. Eye Res. 53 411-414, 1991), 2) CM from cultured human RPE cells(Tombran-Tink et al., Exp. Eye Res. 53 411-414, 1991) and 3) adultbovine IPM prepared as previously described (Tombran-Tink et al., J.Comp. Neurol. 317 175-186, 1992) was performed according to the methodof O'Farrell (J. Biol. Chem. 250 4007-4021, 1975). Isoelectric focusingwas carried out in glass tubes of inner diameter 2.0 mm, using 2% v/vBDH pH 4-8 ampholines for 9,600 volt hours. The final tube gel pHgradient extended from about pH 4.0 to about pH 8.2 as measured by asurface pH electrode. After equilibration for 10 minutes in buffer 0(10% v/v glycerol, 50 mM dithiothreitol, 2.3% w/v SDS and 62.5 mM Tris,pH 6.8), the tube gel was sealed to the top of a 10% w/v acrylamide slabgel (0.75 mm thick) and electrophoresis was carried out for 4 hours at12.5 mAmps/gel. The gels were then either stained with Coomassie blue ortransblotted onto nitrocellulose membrane for Western blot analysis.

Nitrocellulose membranes containing the transblotted proteins werepretreated with 0.5% v/v non-fat dried milk in 50 mM Tris buffer, pH7.4, followed by incubation for 1 hour with an anti-PEDF polyclonalantibody (a gift of Dr. Vincent Cristofalo, Medical College ofPennsylvania) diluted at 1:3,000 in Tris buffer containing 0.5% v/vmilk. After this treatment, the membranes were washed extensively withTris buffer and subjected to further incubation for 30 minutes in anappropriate dilution of alkaline phosphatase-conjugated goat anti-rabbitIgG. The alkaline phosphatase color development reagents p-nitro bluetetrazolium chloride (NBT) and 5-bromo-4-chloro-3′indoyl phosphatep-toluidine salt (BCIP) (BioRad Laboratories, Richmond, Calif.) wereused for the detection of the PEDF protein.

First strand cDNA synthesis of human and monkey RPE cell mRNA wasperformed using the SuperScript Preamplification System catalyzed bySuperScript RNase H-Reverse Transcriptase (RT) (Gibco, BRL,Gaithersburg, Md.). A 3 μl sample of the resulting first strand cDNA wasthen used for each PCR reaction. A pair of primers from the translatedsequences of the PEDF cDNA (Steele et al., Proc. Natl. Acad. Sci. (USA)90 1526-1530, 1993), representing a 1472 bp PCR amplification product,was used in the reactions. The primers were 601 and 499, which have beendescribed previously. The PCR reaction contained 10 mM Tris-HCl, pH 8.3,50 mM KCl, 80 μM of each dNTP, 1 mM MgCl₂, 100 ng of each primer and1.25 units of Amplitag (Perkin Elmer Cetus). A preamplificationdenaturation for 10 minutes at 94° C. was performed and amplificationwas then carried out for 30 cycles. Each cycle was: 94° C., 1 minute;50° C., 1 minute; 72° C., 1 minute. Six μl of the PCR products wereanalyzed on a 1% w/v agarose gel in 1 ×TAE buffer and productsvisualized with ethidium bromide.

Total RNA was isolated by the RNAzol B method (Cinna/Biotecx,Friendswood, Tex.). Ten μg of total RNA was diluted in RNA-loading dyecontaining ethidium bromide (5′Prime-3′Prime, Inc., Boulder, Colo.) andelectrophoresed for 3 hour at 75 V in 1% v/v formaldehyde gels. The RNAwas subsequently transferred onto Nytran membranes (Schleicher &Schuell, Keene, N.H.) using 20×SSC and Stratagene's Posiblotter(Stratagene, La Jolla, Calif.). The membranes were UV crosslinked andbaked at 80° C. for 2 hours prior to hybridization. Fifty ng of a 667 bpPCR amplified product of the PEDF cDNA was used as the probe. This waslabelled with α-³²P-dCTP using a Random Prime It Kit II (Stratagene).Unincorporated nucleotides were removed using Stratagene's Nuctrap Pushcolumns. The membranes were prehybridized for 30 minutes at 68° C. inQuikHyb solution (Stratagene) followed by hybridization for 1 hour at68° C. in the same solution supplemented with 1×10⁶ cpm/ml radiolabelledPEDF probe. Post-hybridization washes were as follows: 2×15 minute at25° C. with 2×SSC/0.1% w/v SDS buffer followed by 1×30 minute at 68° C.with 0.1 ×SSC/0.1% w/v SDS. The hybridized membranes were exposedovernight at −70° using XAR-5 X-Ray films (Eastman Kodak Co. RochesterN.Y.) and intensifying screens.

A human fetal eye of estimated 17 weeks gestation and 1.5 hourspostmortem was fixed in 4% v/v formaldehyde following the removal ofcornea, iris and lens. Following fixation, the posterior segment of theeye was cut into 1×2 mm pieces, dehydrated in ethanol and embedded indiethylene glycol distearate (Polysciences, Warrington, Pa.). Sectionsone micrometer in thickness were cut and handled as previously published(Porrello et al., J. Histochem. Cytochem. 39 171-176, 1991).

A 233 bp fragment containing residues 246-278 of the coding region wasexceed by BamHI and KpnI digestion of human PEDF cDNA (Steele et al.,Proc. Natl. Acad. Sci. (USA) 90 1526-1530, 1993). This fragment was thenligated into pBluescript II KS-phagemid (Stratagene). The phagemid withits 233 bp PEDF insert was then linearized by digestion within itsmultiple cloning site with Styl or with BamHI, neither of which haverestriction sites in the PEDF fragment. T3 and T7 RNA polymerase wereincubated with these DNA templates to generate a sense probe 264 basesand an antisense probe 300 bases in length. Forty of the bases in thesense probe and 67 in the antisense probe were transcribed from vectorDNA.

³⁵S-labeled probes were synthesized with a Riboprobe kit (Promega,Madison, Wis.) according to the manufacturer's instructions. Optimalradiolabel incorporation for a 1 μM volume was typically 1×10⁶ DPM forthe sense and antisense probes. Hybridization and posthybridiation ofthe probes with tissue sections were performed as previously described(Porrello et al., J. Histochem. Cytochem. 39 171-176, 1991) with theexception that the salt washes were performed at 65° C.

Monkey RPE cells, cultured in MEM containing 1% v/v FBS, were seededonto glass coverslips in 24 well plates. The cells were fixed in 4% v/vparaformaldehyde for 15 minutes and non-specific protein binding sitesblocked with 1 mg/ml bovine serum albumin. The PEDF polyclonal antibodywas used at a 1:3,000 dilution and cells were reacted with the antibodyfor 1 hour followed by incubation for 30 minutes in an appropriatedilution of FITC-conjugated goat anti-rabbit IgG. Double-labellingexperiments were performed similarly using an actin monoclonal antibodyand rhodamine-conjugated goat anti-mouse IgG (Pierce, Rockford, Ill.).Immunoreactivity was visualized by epifluorescence microscopy using anOlympus BHS microscope equipped with a 40× UV lens.

Coomassie blue staining of proteins in fetal human RPE-CM shows aprominent 50 kDa PEDF band that was completely absent fromnon-conditioned medium. By Western blot analysis, a comparable band at50 kDa was readily detected by anti-PEDF in conditioned medium from RPEcells and in soluble washes of IPM from a number of species. The 50 kDaprotein was found in RPE-CM from both fetal and adult human RPE cells.CM from young adult monkey (third passage in culture) and from primarychick embryo RPE cells also showed 50 kDa bands with the human PEDFpolyclonal antibody. A positive PEDF band was also detected in solublewashes of adult human IPM and in IPM from fetal and adult cow. PEDF wasoften clearly defined as a doublet in lanes with lower concentration ofPEDF. The presence of PEDF in the IPM indicates that it was secreted invivo in both fetal and young adult where it could influence neuronaldifferentiation and/or survival of retinal neurons.

Interestingly, a 50 kDa protein, was not detected in total cellularextracts of tenth passage RPE cells. To better examine the effect ofcellular ageing, CM from RPE cell up to 15 passages in culture wasexamined. PEDF was found to be secreted in abundance by RPE cells intheir 1st and 2nd passages and at the 3rd passage but only weakly bythese cells in the 5th passage. The protein was not detected in theconditioned medium of RPE cells at the 10th or 15th passages. Thus, PEDFsecretion by RPE cells decreases dramatically in an age-related manner.

Surprisingly, the anti-PEDF antibody detects a doublet migrating at 36kDa rather than at 50 kDa in both the cultured RPE cells and tissueextracts. The low level of the 36 kDa species observed in extracts ofthe neural retina await confirmation by immunocytochemistry in situsince it would be expected that small amounts of RPE contamination wouldbe present in the dissected retinal tissue. This raises the possibilitythat some PEDF is retained intracellularly as a lower molecular weightspecies at least in RPE cells. It is also interesting to note here that,similar to the 50 kDa secreted protein, the 36 kDa species is onlypresent in early passage cells and absent by the loth passage of the RPEcells. Thus, both molecular species disappear in parallel with cellpassages and aging.

By 2-dimensional gel analysis, at least four isoforms of theHPLC-purified native human PEDF protein are detected by Coomassie bluestaining. The spots vary only slightly in apparent molecular weights andthe four most prominent species have apparent pI's of 6.0, 6.2, 6.4 and6.6. The PEDF polyclonal antibody recognizes all four isoforms of thisprotein and possibly two others in both human RPE-CM and bovine IPM asshown by Western blot analysis of the transblotted 2-D gels.

Using primers 601 and 499 which encompass a 1.47 kb sequence of the PEDFmessage, a single product of approximately 1.5 kb is amplified in bothfetal human and adult monkey RPE cells. By Northern blot analysis, a 1.5kb band is also detected with the 667 bp PCR-amplified PEDF probe incultures of fetal and adult human RPE cells as well as in RPE cellexplants. Relatively more PEDF mRNA is seen in the fetal RPE cellexplants as compared to cultured cells. No difference is seen in thesize of the messages under any of the conditions (fetal vs. adult, humanvs. monkey). Thus, by Western and Northern blot analyses and by PCR, themolecular size and apparent structure of the 50 kDa protein and itsmessage appears to be similar in human, monkey and bovine RPE cells,retina and IPM.

Five μg of total RNA from monkey retina and from 1st and 10th passagemonkey RPE cells were electrophoresed and a weak hybridization signalwas seen from total retina RNA. A more, prominent 1.5 kb transcript bandwas present in the early passage (1st passage) monkey RPE cells while notranscript is detected in the late (10th) passaged RPE cells. Thus,similar to the secretion pattern of PEDF protein after successive RPEcell passages, late passage monkey RPE cells do not transcribe the PEDFmRNA.

The effect of PEDF on Y79 retinoblastoma cell line differentiation wasfirst demonstrated with conditioned media from human fetal RPE cells.Thus, we performed in situ hybridization of a fetal human retina of adevelopmental age similar to that of the cells used for fetal human RPEculture (17 weeks gestation). Retinas hybridized with antisense probesunder high stringency (65° C.) showed specific binding of the probe tothe RPE cell layer. The retinal neuroblastic layer which containsincompletely differentiated neurons and glia and the ganglion celllayer, which was well differentiated at this developmental stage, werenot labeled above background.

In well attached cells (3rd passage, 5-10 days postattachment),immunofluorescence was seen in association with cytoskeletal strandswithin the cells and within the nucleus. At 2 days of attachment, theprotein was concentrated around the nucleus and was of a granularnature. Staining was also associated with a fibrous cytoskeletal-likenetwork. When monkey RPE cells are examined soon after seeding (1-2hours), discrete localization of the PEDF protein is seen at the tips ofmany processes of the RPE cell. Similar to the dramatic decrease seen inPEDF secretion and transcription with successive cell passages, noimmunoreactivity with the PEDF polyclonal antibody is seen in culturedmonkey RPE cells after the tenth passage.

To further examine the association of PEDF immunoreactivity withcytoskeletal elements within the cytoplasm, we analyzed the structuresby double-labelling using anti-PEDF as described above and an actinantibody detected by rodamine-conjugated goat anti-mouse IgG. Bothantibodies localized to structures around the nucleus and tocytoskeletal strands in the cytoplasm. Concentration of both antibodiesis also seen at the tip of a pseudopod. Because of PEDF's apparentassociation with cytoskeletal structures, possibly actin microfilaments,we addressed the possibility of antibody cross reactivity with actin.Anti-PEDF did not bind to purified actin on a Western blot. Only apositive PEDF band at 50 kDa is observed in RPE conditioned medium. Theantibody also does not recognize human α-1-antitrypsin, a homologousmember of the serpin supergene family. It thus seems that the PEDFantibody specifically recognizes and binds to PEDF (50 and/or 36 kDaforms) associated with the intracellular actin cytoskeletal network ofthe RPE cells but does not bind directly to purified actin on a Westernblot.

EXAMPLE 56 PEDF as a Survival Factor for Cerebellar Granule Cells inCulture

Cerebellar granule cells (CGC) were prepared from 5 or 8-day-old SpragueDawley rat pups (Taconic Farms) as previously described (Novelli et al.,Brain Res. 451 205-212, 1988; Levi et al., In: A Dissection and TissueCulture Manual of the Nervous System (Shahar, de Vellis, Vernadakis andHaber, eds), pp. 211-214. Alan R. Liss, Inc. New York. 1989). In brief,tissue free of meninges was minced in a buffer containing 124 mM NaCl, 1mM NaH₂PO₄, 1.2 mM MgSO₄, 3 mg/ml bovine serum albumin (BSA), 27 μMphenol red, and 25 mM HEPES, pH 7.4, and centrifuged at 550×g for 3minutes. The tissue pellet from 10-20 animals was resuspended andtrypsinized for 15 minutes at 37° C. in 30 ml of the same buffercontaining 250 μg/ml trypsin; a further 15 ml of buffer, containing 26μg/ml DNase I (from Sigma Chemical Co.), 166 μg/ml soybean trypsininhibitor (from Sigma Chemical Co.) and 0.5 mM additional MgSO₄, wereadded and the tissue was centrifuged again as described above. Thepellet was resuspended in 1 ml of buffer supplemented with 80 μg/mlDNase, 0.52 mg/ml of trypsin inhibitor, and 1.6 mM additional MgSO₄, andtriturated 60 times with a Pasteur pipette. The suspension was dilutedwith 2 ml of buffer containing 0.1 mM CaCl₂ and 1.3 mM additional MgSO₄,and undissociated material was allowed to settle for 5 minutes. Thesupernatant was transferred to another tube, cells were recovered bybrief centrifugation and resuspended in serum-containing medium (Eagle'sbasal medium, from GIBCO, with 25 mM KCl, 2 mM glutamine, from GIBCO,100 μg/ml gentamicin, and 10% v/v heat-inactivated fetal calf serum,from GIBCO) or chemically defined medium (DMEM:F12 (1:1, from GIBCO)with 5 μg/ml insulin (from Boehringer Mannheim), 30 nM selenium (fromBoehringer Mannheim), 100 μg/ml transferrin (from GIBCO), 100 nMputrescine (from Sigma Chemical Co.), 20 nM progesterone (from SigmaChemical Co.), 50 U/ml penicillin, 50 μg/ml streptomycin, and 2 nMglutamine; Bottenstein, In: Cell Culture in the Neurosciences,Bottenstein and Sato, eds., pp. 3-43. Plenum Publishing Corp, New York,1985). Cells were plated in poly-L-lysine-coated 96-well plates (for MTSassay, MTS assay kit were obtained from Promega Corp., and neurofilamentELISA assay) or 8-well chamber slides (for immunocytochemistry and5-bromo-2′-deoxyuridine, BrdU, labelling) at 2.5×10⁵ cells/cm²and grownat 37° C. in a humidified atmosphere comprising 5% v/v CO₂ in air. After1 day in culture, cytosine arabinoside (Ara-C) was added only to cellsin serum-containing medium to a final concentration of 10 μM.

Cerebellar granule cells in 96-well plates were incubated in a CO₂incubator for 4 hours with MTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4--sulfophenyl)-2H-tetrazolium,inner salt) and PMS (phenazine methosulfate) at final concentration of333 μg/ml MTS and 25 μM PMS, respectively. In the presence of PMS, MTSis converted to a water-soluble formazan by a dehydrogenase enzyme foundin metabolically active cells (Cory et al., Cancer Commun. 3 207-212,1991). The quantity of formazan product was determined byspectrophotometry (Dynatech microplate reader) at 490 nm.

After 7 days in vitro (DIV), the cells were washed three times incalcium- and magnesium-free phosphate-buffered saline (PBS) and fixedwith 2% v/v paraformaldehyde for 10 minutes, followed by 10 minutes at−20° C. in 95% v/v ethanol/5% v/v acetic acid. Incubation with primaryantibodies against NSE (neuron-specific enolasecalbindin, from SigmaChemical Co.), GABA (from Sigma Chemical Co.), calbindin (from SigmaChemical Co.), or glial fibrillary acidic protein (GFAP) was carried outfor 60 minutes at room temperature. Antibodies were applied at dilutionsof 1:500-1:5,000 in the presence of 2% v/v normal goat serum and 0.2%v/v BSA. The antibodies were visualized using the ABC system (fromVector Lab) and diaminobenzidine. At least 20 fields were counted from2-3 wells for each experiment. The average number of cells per field wasthen calculated to determine the ratio for the number of cells stainedby the other antibodies relative to NSE-positive cells in controlcultures.

BrdU labeling (BrdU labeling kit was supplied by—Amersham) was performedby the method described (Gratzner, Science 218 474-475, 1982; Gao etal., Neuron 6, 705-715, 1991) with the following modifications. Thecells were plated in 8-well chamber slides and PEDF was immediatelyadded. For this experiment, DMEM rather than DMEM:F12 was used inchemically defined medium (CDM) and no Ara-C was added toserum-containing medium (SCM). After 24 hours, BrdU (diluted 1:100;Amersham cell proliferation kit) was added to the culture medium for 24hours, after which the cells were fixed in 2% v/v paraformaldehyde for10 minutes, treated with 95% v/v ethanol, 15% v/v acetic acid for 10minutes, and incubated with an anti-BrdU monoclonal antibody, which wasdiluted 1:20, for 2 hours. The cultures were then incubated with ahorseradish peroxidase-conjugated goat anti-mouse secondary antibody(Vector Lab) for 60 minutes. After incubation with diaminobenzidine, thecells were mounted in Gel Mount. The mitotic index was determined bycounting the percentage of labeled cells with microscopy. For eachvalue, a random sample of 3,000 cells was counted.

Neurofilament ELISA was performed according to the method of Doherty etal. (J. Neurochem 42 1116-1122, 1984) with slight modifications.Cultures grown in 96-well microtiter plates were fixed with 4% v/vparaformaldehyde in PBS at 4° C. for 2 hours. The fixed cells werepermeabilized by treatment for 15 minutes with 0.1% v/v Triton X-100 inPBS, followed by incubation for 60 minutes with PBS containing 10% v/vgoat serum to block nonspecific binding. The cultures were thenincubated with a monoclonal anti-neurofilament antibody (RMO-42)overnight at 4° C. at a dilution of 1:100 (Lee et al., Proc. Natl. Acad.Sci. USA 85 7384-7388, 1988). After washing twice with PBS containing10% v/v goat serum, cells were incubated with secondary antibody(horseradish peroxidase-conjugated goat anti-mouse at a dilution of1:1,000) for 1 hour. Following sequential washing with PBS and water,the cultures were incubated with 0.2% v/v o-phenylenediamine (OPD, fromSigma Chemical Co.) and 0.02% v/v H₂O₂ in 50 mM citrate buffer, pH 5.0,for 30 minutes. The reaction was stopped by adding an equal volume of4.5 M H₂SO₄. Product formation was quantitated by reading the opticaldensity (O.D.) of an aliquot of the reaction product at 490 nm using aDynatech microplate reader.

The PEDF used in the experiments was a recombinant construct(Asp⁴⁴-Pro⁴¹⁸) as previously described (Becerra et al., J. Biol. Chem.268 23148-23156, 1993) and as described above. The recombinantexpression construct from the human PEDF cDNA was expressed inEscherichia coli and purified from the bacterial inclusion bodies. rPEDFwas highly purified by gel filtration and cation exchangechromatography, and demonstrated neurotrophic activity on humanretinoblastoma Y-79 cells, as reported for the native PEDF (Becerra etal., J. Biol. Chem. 268 23148-23156, 1993) and as described above. rPEDFwas added in urea at a final concentration of 500 ng/ml urea and 10 mMrPEDF. All controls contained the concentration of urea corresponding tothat present in the rPEDF added and no dose of urea affected any of theassays.

All experiments were replicated twice. The statistical analyses thatwere carried out by standard statistical methods.

Unless indicated otherwise, all experiments were carried out using CGCprepared from postnatal day 8 (P8) rats. In SCM, optical density (O.D.)was proportional to the cell number plated over a range from 1 to 9×10⁵cells/cm². In contrast, for cells grown CDM, the linear range covered 1to 5×10⁵ cells/cm². For all subsequent experiments, cells were plated at2.5×10⁵ cells/cm², i.e., within the linear range for either type ofculture medium.

Exposure of cells to 500 ng/ml (equivalent to 11.68 nM) rPEDF in SCMresulted in a statistically significant difference in the O.D. betweenrPEDF-treated and untreated cultures by two days in vitro (DIV 2): thedifference lasted up to DIV 10. By DIV 4, there was a 50% increase incell number in the presence of rPEDF, with a 30-40% increase maintainedthrough DIV 10. A dose-response curve demonstrated that the effect ofrPEDF was statistically significant at 500 ng/ml or above, althoughincreasing the concentration above 500 ng/ml did not produce furtherincreases. PEDF had a much larger effect on cell number when added toCGC in chemically defined medium. The time course of the rPEDF effect inCDM. The time course analysis demonstrates that rPEDF-treated culturescontain significantly more cells than control cultures by DIV 4, witheven larger differences by DIV 7 and 10. Most of the 2-3 fold differencewas the result of large decreases in cell numbers in the controlcultures. The dose-response curve in CDM shows that there is astatistically significant effect at 20 ng/ml (equivalent to 0.47 nM)rPEDF, 25-fold lower than the effective dose in serum-containing medium.Increasing the concentration of rPEDF above 50 ng/ml did not promotefurther survival of CGC in CDM.

Immunocytochemistry was used to identify the cell types present in CGCcultures before and after treatment with rPEDF. Postnatal day 8 CGCcultures grown for 7 days with or without rPEDF (500 ng/ml) were stainedwith four different antibodies: 1) a polyclonal rabbit antibody toneuron-specific enolase (NSE), which recognizes all differentiatedcerebellar neurons, including P8 CGC which are differentiated bothmorphologically and pharmacologically by DIV 7; 2) a polyclonal antibodyto GABA, which stains all cerebellar neurons except cerebellar granulecells; 3) an antibody to calbindin, which is specific for Purkinjecells; 4) an antibody to glial fibrillary acidic protein (GFAP), anintermediate filament protein present only in astrocytes. The resultsare summarized in Table IV.

TABLE IV Antigen Treatment SCM CDM NSE Control 100.0 ± 6.2 100.0 ± 4.5PEDF 127.0 ± 5.9* 157.2 ± 7.4* GABA Control 2.8 ± 0.2 1.4 ± 0.2 PEDF 3.2± 0.2 1.8 ± 0.2 Calbindin Control 0.06 ± 0.01 0.07 ± 0.02 PEDF 0.07 ±0.02 0.12 ± 0.02 GFAP Control 0.86 ± 0.07 0.99 ± 0.07 PEDF 0.06 ± 0.03*0.60 ± 0.06* *p < 0.005 relative to the respective control

There were significantly more NSE-positive cells in both SCM (30%increase) and in CDM (60% increase) in the presence of rPEDF. Nostatistically significant increase was observed in the number ofGABA-positive neurons or Purkinje cells (calbindin-positive).Furthermore, the results support the purity of the CGC cultures, sinceonly 1-3% of the cells are GABA-positive non-CGC neurons, less than 0.1%are Purkinje cells and less than 1% are astrocytes. The increased numberof NSE-positive cells in the presence of rPEDF, therefore, reflectsenhanced survival of CGC neurons.

In order to determine whether the increase in O.D. (MTS assay) inresponse to PEDF reflected simply cell survival or an increase inproliferation, a BrdU labeling study was performed using cultures frompostnatal day 5 (P5) animals, a time when cerebellar granule cells arestill dividing in vivo. First, the effect of rPEDF on P5 CGC cultures atDIV 1 and 2 was determined using the MTS assay. PEDF had no effect atDIV 1 but caused a small but significant increase in O.D. at DIV 2 inboth serum-containing medium and chemically defined medium. Therefore,BrdU was added on DIV 1 and cells were fixed on DIV 2. Under controlconditions, the BrdU labeling index was 5% in SCM and 3% in CDM: PEDFdid not increase the BrdU labeling index in either culture medium. Thelack of effect of PEDF on BrdU labeling showed that enhanced survivalrather than increased cell division underlies the larger cell numberseen with PEDF.

In order to investigate the effects of rPEDF on neurite outgrowth fromP8 CGC, a neurofilament ELISA assay was used. Immunocytochemistry hadshown that the monoclonal antibody RMO-42, which recognizes thephosphorylated domain of NF-H (200 kDa) and NF-M (160 kDa), stained onlythe neurites of cerebellar granule cells in culture in comparison toanti-NSE which stained both neurites and cell bodies. Therefore, thisantibody was used as a direct measure of neurofilament protein presentin the neurites and an indirect measure of number of neurites. Using theneurofilament ELISA, the O.D. was found to be proportional to the numberof cells originally plated when the assay was carried out on DIV 7, atime when all cells have developed a full complement of processes. rPEDFincreased neurofilament content, both in SCM (45%) and CDM (43%) but theincrease was directly proportional to the increase in cell number. Whenthe data are expressed as the increase in neurofilament relative to theincrease in cell number, these ratios are about 1.0 for both SCM andCDM. These results demonstrated that PEDF does not promote neuriteoutgrowth from CGC in culture. Microscopic observation revealed nomorphological change caused by rPEDF.

In the present study, we show that PEDF increased the number of viablecells in cerebellar granule cell cultures, whether the cells wereprepared from postnatal day 5 or postnatal day 8 rats, and whether thecultures were maintained in serum-containing medium or chemicallydefined medium. In SCM, PEDF showed no effect at DIV 1, but by DIV 4,about 50% more cells were present in PEDF-containing medium, adifference which was maintained through DIV 10. In CDM, a similar 50%difference was seen by DIV 4, with the difference becoming larger at DIV7 and DIV 10 (final 2-3 fold more cells at these times in the presenceof PEDF). The effect of PEDF showed a dose-response relationship in bothSCM and CDM. However, whereas a significant effect was seen with 500ng/ml (equivalent to 11.68 nM) of rPEDF in SCM, only 20 ng/ml (0.47 nM)of PEDF were required to achieve a significant increase in CDM. Thus,effects of PEDF were larger and occurred at 25-fold lower doses in CDMthan in SCM. Furthermore, these effects of rPEDF were long-lasting inthat only a single dose of the factor was added to culture medium at thestart of the culture period. The relatively high potency of PEDF in CDMcompared to SCM may be due, at least in part, to the presence ofinhibitors or degradative enzymes in the fetal calf serum that reducedthe bioactivity of PEDF. Alternatively, another trophic factor, additivein action to PEDF, may be present in serum, which masks the fullbiological activity of PEDF.

To determine whether this PEDF-induced difference in cell numberreflected an increase in cell survival or an increase in proliferation,BrdU incorporation was measured in the presence of PEDF using cells frompostnatal day 5 animals, an age when cerebellar granule cells are stilldividing in vivo. PEDF did not alter BrdU incorporation into cellscultured in either SCM or CDM. The lack of effect of PEDF on BrdUincorporation indicates that enhanced survival rather than increasedcell division underlies the larger cell number seen with PEDF. Since theMTS assay only determines total cell number, immunocytochemistry wasused to identify the general types and relative proportions of cellspresent in cultures treated with PEDF. There were significantly moreNSE-positive cells in PEDF-treated cultures. However, no statisticallysignificant effect of PEDF was found on GABA-positive cells, whichincludes all cerebellar neurons except the granule cells, or on thenumber of calbindin-positive Purkinje cells. Since GABA-positive cellsrepresent only 1-3% of the total NSE-positive neurons in the culturesand even fewer calbindin-positive cells are present (about 0.1%), itthus appears that PEDF affects only the CGC neurons. Moreover, theeffect seems to be on long-term neuronal survival since PEDF does notpromote increased mitotic activity. Our present results are ofparticular interest with regard to recent results on the expression ofthe PEDF gene in WI-38 fibroblast cells in culture. In these cells, PEDF(called EPC-1) mRNA accumulates in young WI-38 cells at Go but little orno transcription is found in senescent cells. No PEDF mRNA has beendetected in P8 CGC neurons, in agreement with their lack of mitosis byP8. Just what roles PEDF may play in cell cycle or survival eventsremain to be determined. In this regard, it will be interesting in thefuture to determine if PEDF exerts an anti-apoptotic effect in promotingthe survival of CGC.

Both purified native PEDF and recombinant PEDF exhibit a strikingneurotrophic effect on cultured retinoblastoma cells as outlined above.rPEDF had no comparable effect on the morphology of the CGC, however. Toinvestigate a possible effect of PEDF on neurite outgrowth, we used aspecific neurofilament assay designed to quantify neurite outgrowth.Although the neurofilament content was higher at DIV 7 in PEDF-treatedcultures (40% in both SCM and CDM), this simply reflected the fact thatthere were more cells present in the PEDF-treated cultures (30% in SCMand 60% in CDM by immunocytochemistry). The biochemical data are thus inconcert with the morphological data and indicate that PEDF has no effecton neurite outgrowth in CGC.

Since PEDF shares considerable sequence homology with members of theserine protease inhibitor (serpin) supergene family, PEDF may act byinhibiting protease activity. The glia-derived nexins, other members ofthe serpin family, have been proposed to exert neurotrophic actions byregulating the balance between protease and protease inhibitoractivities. However, PEDF lacks significant homology to the proposedconsensus sequence for the serpin reactive center region, which itselfis heterogeneous in length and amino acid composition. Furthermore,protease inhibition assays showed that PEDF did not affect trypsin,chymotrypsin, elastase, cathepsin G, endoproteinase Lys-C,endoproteinase Glu-C, or subtilisin activity, suggesting that inhibitionof known serine proteinase may not be the biochemical pathway for thePEDF neurotrophic activity.

Aside from a direct action on cerebellar granule cells, it is alsopossible that PEDF acts to regulate expression of some otherneurotrophic factor. BDNF and NT-3 are both present in the immaturecerebellum, and Trk B, the BDNF receptor, is also expressed in thecerebellum early in development. BDNF was shown to act as a survivalfactor for embryonic granule cells. It increased cerebellar granule cellnumber 50 to 100% at 20 ng/ml, with the effect occurring after DIV 4,results very comparable to ours with PEDF. Furthermore, BDNF has noeffect on mitosis of cerebellar granule cells. Since a number ofhormones and neurotransmitters are known to affect the production ofneurotrophins by neurons, the effect of PEDF could be mediated throughregulation of one of these factors. Tri-iodothyronine, for example,enhanced the production of NT-3 in CGC cultures while neurotransmittersor analogs such as kainic acid and GABA increased the synthesis of NGFand BDNF in hippocampal neurons. Thus, PEDF may act directly through itsown receptor and signal transduction pathway, or more indirectly throughregulation of the production of other neurotrophic factors in the CGCcultures.

All of the references cited herein are hereby incorporated in theirentireties by reference.

The above descriptions of exemplary embodiments of retinal pigmentedepithelium derived neurotrophic factor are for illustrative purposes.Because of variations which will be apparent to those skilled in theart, the present invention is not intended to be limited to theparticular embodiments described above. The present invention may alsobe practiced in the absence of any element not specifically disclosed.The scope of the invention is defined by the following claims.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 34 <210> SEQ ID NO 1 <211>LENGTH: 1503 <212> TYPE: DNA <213> ORGANISM: HUMAN <220> FEATURE: <221>NAME/KEY: mRNA <222> LOCATION: (1)..(1503) <221> NAME/KEY: CDS <222>LOCATION: (117)..(1373) <223> OTHER INFORMATION: PRODUCT = PIGMENTEPITHELIAL-DERIVED FACTOR GENE = PEDF CODON_START =1 <221> NAME/KEY:sig_peptide <222> LOCATION: (117)..(170) <223> OTHER INFORMATION: GENE =pedf CODON_START = 1 <221> NAME/KEY: mat_peptide <222> LOCATION:(171)..(1370) <223> OTHER INFORMATION: PRODUCT = PIGMENTEPITHELIAL-DERIVED FACTOR GENE = PEDF CODON_START = 1 <400> SEQUENCE: 1ggacgctgga ttagaaggca gcaaaaaaag atctgtgctg gctggagccc cctcagtgtg 60caggcttaga gggactaggc tgggtgtgga gctgcagcgt atccacaggc cccagg atg 119Met cag gcc ctg gtg cta ctc ctc tgc att gga gcc ctc ctc ggg cac agc 167Gln Ala Leu Val Leu Leu Leu Cys Ile Gly Ala Leu Leu Gly His Ser -15 -10-5 agg tgc cag aac cct gcc agc ccc ccg gag gag ggc tcc cca gac ccc 215Arg Cys Gln Asn Pro Ala Ser Pro Pro Glu Glu Gly Ser Pro Asp Pro -1 1 510 15 gac agc aca ggg gcg ctg gtg gag gag gag gat cct ttc ttc aaa gtc263 Asp Ser Thr Gly Ala Leu Val Glu Glu Glu Asp Pro Phe Phe Lys Val 2025 30 ccc gtg aac aag ctg gca gcc gct gtc tcc aac ttc ggc tat gac ctg311 Pro Val Asn Lys Leu Ala Ala Ala Val Ser Asn Phe Gly Tyr Asp Leu 3540 45 tac cgg gtg cga tcc agg atg agc ccc acg acc aac gtg ctc ctg tct359 Tyr Arg Val Arg Ser Arg Met Ser Pro Thr Thr Asn Val Leu Leu Ser 5055 60 cct ctc agt gtg gcc acg gcc ctc tcg gcc ctc tcg ctg gga gcg gag407 Pro Leu Ser Val Ala Thr Ala Leu Ser Ala Leu Ser Leu Gly Ala Glu 6570 75 cag cga aca gaa tcc atc att cac cgg gct ctc tac tat gac ttg atc455 Gln Arg Thr Glu Ser Ile Ile His Arg Ala Leu Tyr Tyr Asp Leu Ile 8085 90 95 agc agc cca gac atc cat ggt acc tat aag gag ctc ctt gac acg gtc503 Ser Ser Pro Asp Ile His Gly Thr Tyr Lys Glu Leu Leu Asp Thr Val 100105 110 act gcc ccc cag aag aac ctc aag agt gcc tcc cgg atc gtc ttt gag551 Thr Ala Pro Gln Lys Asn Leu Lys Ser Ala Ser Arg Ile Val Phe Glu 115120 125 aag aag cta cgc ata aaa tcc agc ttt gtg gca cct ctg gaa aag tca599 Lys Lys Leu Arg Ile Lys Ser Ser Phe Val Ala Pro Leu Glu Lys Ser 130135 140 tat ggg acc agg ccc aga gtc ctg acg ggc aac cct cgc ttg gac ctg647 Tyr Gly Thr Arg Pro Arg Val Leu Thr Gly Asn Pro Arg Leu Asp Leu 145150 155 caa gag atc aac aac tgg gtg cag gcg cag atg aaa ggg aag ctc gcc695 Gln Glu Ile Asn Asn Trp Val Gln Ala Gln Met Lys Gly Lys Leu Ala 160165 170 175 agg tcc aca aag gaa att ccc gat gag atc agc att ctc ctt ctcggt 743 Arg Ser Thr Lys Glu Ile Pro Asp Glu Ile Ser Ile Leu Leu Leu Gly180 185 190 gtg gcc cac ttc aag ggg cac tcc gta aca aag ttt gac tcc agaaag 791 Val Ala His Phe Lys Gly His Ser Val Thr Lys Phe Asp Ser Arg Lys195 200 205 act tcc ctc gag gat ttc tac ttg gat gaa gag agg acc gtg agggtc 839 Thr Ser Leu Glu Asp Phe Tyr Leu Asp Glu Glu Arg Thr Val Arg Val210 215 220 ccc atg atg tcg gac cct aag gct gtt tta cgc tat ggc ttg gattca 887 Pro Met Met Ser Asp Pro Lys Ala Val Leu Arg Tyr Gly Leu Asp Ser225 230 235 gat ctc agc tgc aag att gcc cag ctg ccc ttg acc gga agg atgagt 935 Asp Leu Ser Cys Lys Ile Ala Gln Leu Pro Leu Thr Gly Arg Met Ser240 245 250 255 atc atc ttc ttc ctg ccc ctg aaa gtg acc cag aat ttg accttg ata 983 Ile Ile Phe Phe Leu Pro Leu Lys Val Thr Gln Asn Leu Thr LeuIle 260 265 270 gag gag agc ctc acc tcc gag ttc att cat gac ata gac cgagaa ctg 1031 Glu Glu Ser Leu Thr Ser Glu Phe Ile His Asp Ile Asp Arg GluLeu 275 280 285 aag acc gtg cag gcg gtc ctc act gtc ccc aag ctg aag ctgagt tac 1079 Lys Thr Val Gln Ala Val Leu Thr Val Pro Lys Leu Lys Leu SerTyr 290 295 300 gaa ggc gaa gtc acc aag tcc ctg cag gag atg aag ctg caatcc ttg 1127 Glu Gly Glu Val Thr Lys Ser Leu Gln Glu Met Lys Leu Gln SerLeu 305 310 315 ttt gat tca cca gac ttt agc aag atc aca ggc aaa ccc atcaag ctg 1175 Phe Asp Ser Pro Asp Phe Ser Lys Ile Thr Gly Lys Pro Ile LysLeu 320 325 330 335 act cag gtg gaa cac cgg gct ggc ttt gag tgg aac gaggat ggg gcg 1223 Thr Gln Val Glu His Arg Ala Gly Phe Glu Trp Asn Glu AspGly Ala 340 345 350 gga acc acc ccc agc cca ggg ctg cag cct gcc cac ctcacc ttc ccg 1271 Gly Thr Thr Pro Ser Pro Gly Leu Gln Pro Ala His Leu ThrPhe Pro 355 360 365 ctg gac tat cac ctt aac cag cct ttc atc ttc gta ctgagg gac aca 1319 Leu Asp Tyr His Leu Asn Gln Pro Phe Ile Phe Val Leu ArgAsp Thr 370 375 380 gac aca ggg gcc ctt ctc ttc att ggc aag att ctg gacccc agg ggc 1367 Asp Thr Gly Ala Leu Leu Phe Ile Gly Lys Ile Leu Asp ProArg Gly 385 390 395 ccc taa tatcccagtt taatattcca ataccctaga agaaaacccgagggacagca 1423 Pro 400 gattccacag gacacgaagg ctgcccctgt aaggtttcaatgcatacaat aaaagagctt 1483 tatccctaaa aaaaaaaaaa 1503 <210> SEQ ID NO 2<211> LENGTH: 418 <212> TYPE: PRT <213> ORGANISM: HUMAN <400> SEQUENCE:2 Met Gln Ala Leu Val Leu Leu Leu Cys Ile Gly Ala Leu Leu Gly His 1 5 1015 Ser Arg Cys Gln Asn Pro Ala Ser Pro Pro Glu Glu Gly Ser Pro Asp 20 2530 Pro Asp Ser Thr Gly Ala Leu Val Glu Glu Glu Asp Pro Phe Phe Lys 35 4045 Val Pro Val Asn Lys Leu Ala Ala Ala Val Ser Asn Phe Gly Tyr Asp 50 5560 Leu Tyr Arg Val Arg Ser Arg Met Ser Pro Thr Thr Asn Val Leu Leu 65 7075 80 Ser Pro Leu Ser Val Ala Thr Ala Leu Ser Ala Leu Ser Leu Gly Ala 8590 95 Glu Gln Arg Thr Glu Ser Ile Ile His Arg Ala Leu Tyr Tyr Asp Leu100 105 110 Ile Ser Ser Pro Asp Ile His Gly Thr Tyr Lys Glu Leu Leu AspThr 115 120 125 Val Thr Ala Pro Gln Lys Asn Leu Lys Ser Ala Ser Arg IleVal Phe 130 135 140 Glu Lys Lys Leu Arg Ile Lys Ser Ser Phe Val Ala ProLeu Glu Lys 145 150 155 160 Ser Tyr Gly Thr Arg Pro Arg Val Leu Thr GlyAsn Pro Arg Leu Asp 165 170 175 Leu Gln Glu Ile Asn Asn Trp Val Gln AlaGln Met Lys Gly Lys Leu 180 185 190 Ala Arg Ser Thr Lys Glu Ile Pro AspGlu Ile Ser Ile Leu Leu Leu 195 200 205 Gly Val Ala His Phe Lys Gly HisSer Val Thr Lys Phe Asp Ser Arg 210 215 220 Lys Thr Ser Leu Glu Asp PheTyr Leu Asp Glu Glu Arg Thr Val Arg 225 230 235 240 Val Pro Met Met SerAsp Pro Lys Ala Val Leu Arg Tyr Gly Leu Asp 245 250 255 Ser Asp Leu SerCys Lys Ile Ala Gln Leu Pro Leu Thr Gly Arg Met 260 265 270 Ser Ile IlePhe Phe Leu Pro Leu Lys Val Thr Gln Asn Leu Thr Leu 275 280 285 Ile GluGlu Ser Leu Thr Ser Glu Phe Ile His Asp Ile Asp Arg Glu 290 295 300 LeuLys Thr Val Gln Ala Val Leu Thr Val Pro Lys Leu Lys Leu Ser 305 310 315320 Tyr Glu Gly Glu Val Thr Lys Ser Leu Gln Glu Met Lys Leu Gln Ser 325330 335 Leu Phe Asp Ser Pro Asp Phe Ser Lys Ile Thr Gly Lys Pro Ile Lys340 345 350 Leu Thr Gln Val Glu His Arg Ala Gly Phe Glu Trp Asn Glu AspGly 355 360 365 Ala Gly Thr Thr Pro Ser Pro Gly Leu Gln Pro Ala His LeuThr Phe 370 375 380 Pro Leu Asp Tyr His Leu Asn Gln Pro Phe Ile Phe ValLeu Arg Asp 385 390 395 400 Thr Asp Thr Gly Ala Leu Leu Phe Ile Gly LysIle Leu Asp Pro Arg 405 410 415 Gly Pro <210> SEQ ID NO 3 <211> LENGTH:379 <212> TYPE: PRT <213> ORGANISM: HUMAN <220> FEATURE: <223> OTHERINFORMATION: /note= Met 1...Ile 4 is an N-terminal fusion to Asp44...Pro 418 of SEQ ID No: 2; Met 1...Glu 43 of SEQ ID NO:2 is deleted<400> SEQUENCE: 3 Met Asn Arg Ile Asp Phe Phe Phe Lys Val Pro Val AsnLys Leu Ala 1 5 10 15 Ala Ala Val Ser Asn Phe Gly Lys Asp Leu Tyr ArgVal Arg Ser Ser 20 25 30 Met Ser Pro Thr Thr Asn Val Leu Leu Ser Pro LeuSer Val Ala Thr 35 40 45 Ala Leu Ser Ala Leu Ser Ser Gly Ala Glu Gln ArgThr Glu Ser Ile 50 55 60 Ile His Arg Ala Leu Tyr Tyr Asp Leu Ile Ser SerPro Asp Ile His 65 70 75 80 Gly Thr Tyr Lys Glu Leu Leu Asp Thr Val ThrAla Pro Gln Lys Asn 85 90 95 Leu Lys Ser Ala Ser Arg Ile Val Phe Glu LysLys Leu Arg Ile Lys 100 105 110 Ser Ser Phe Val Ala Pro Leu Glu Lys SerVal Gly Thr Arg Pro Arg 115 120 125 Val Leu Thr Gly Asn Pro Arg Leu AspLeu Gln Glu Ile Asn Asn Trp 130 135 140 Val Gln Ala Gln Met Lys Gly LysLeu Ala Arg Ser Thr Lys Gln Ile 145 150 155 160 Pro Asp Glu Ile Ser IleLeu Leu Leu Gly Val Ala His Phe Lys Gly 165 170 175 Gln Trp Val Thr LysPhe Asp Ser Arg Lys Thr Ser Leu Glu Asp Phe 180 185 190 Tyr Leu Asp GluGlu Arg Thr Val Arg Val Pro Met Met Ser Asp Pro 195 200 205 Lys Ala ValLeu Arg Tyr Gly Leu Asp Ser Asp Leu Ser Cys Lys Ile 210 215 220 Ala GlnLeu Pro Leu Thr Gly Ser Met Ser Ile Ile Phe Phe Leu Pro 225 230 235 240Leu Lys Val Thr Gln Asn Leu Thr Leu Ile Glu Glu Ser Leu Thr Ser 245 250255 Glu Phe Ile His Asp Ile Asp Arg Glu Leu Lys Thr Val Gln Ala Val 260265 270 Leu Thr Val Pro Lys Leu Lys Leu Ser Tyr Glu Gly Glu Val Thr Lys275 280 285 Ser Leu Gln Glu Met Lys Leu Gln Ser Leu Phe Asp Ser Pro AspPhe 290 295 300 Ser Lys Ile Thr Gly Lys Pro Ile Lys Leu Thr Gln Val GluHis Arg 305 310 315 320 Ala Gly Phe Glu Trp Asn Glu Asp Gly Ala Gly ThrThr Pro Ser Pro 325 330 335 Gly Leu Gln Pro Ala His Leu Thr Phe Pro LeuAsp Tyr His Leu Asn 340 345 350 Gln Pro Phe Ile Phe Val Leu Arg Asp ThrAsp Thr Gly Ala Leu Leu 355 360 365 Phe Ile Gly Lys Ile Leu Asp Pro ArgGly Pro 370 375 <210> SEQ ID NO 4 <211> LENGTH: 14581 <212> TYPE: DNA<213> ORGANISM: HUMAN <220> FEATURE: <223> OTHER INFORMATION: mRNA:6683; EXON: 6683-6790; EXON 11584- 11675; EXON: 14539-14581; INTRON:6791-11583; INTRON: 11676-14538; CDS: 11584-11675; 14539-14580 <400>SEQUENCE: 4 gatctagagc ggccgcaggg tggactgtgc tgaggaaccc tgggcccagcagggtggcag 60 cccgcgcagt gccacgtttg gcctctggcc gctcgccagg catcctccaccccgtggtcc 120 cctctgacct cgccagccct cccccgggac acctccacgc cagcctggctctgctcctgg 180 cttcttcttc tctctatgcc tcaggcagcc ggcaacaggg cggctcagaacagcgccagc 240 ctcctggttt gggagaagaa ctggcaatta gggagtttgt ggagcttctaattacacacc 300 agcccctctg ccaggagctg gtgcccgcca gccgggggca ggctgccgggagtacccagc 360 tccagctgga gacagtcagt gcctgaggat ttgggggaag caggtggggaaaccttggca 420 cagggctgac accttcctct gtgccagagc ccaggagctg gggcagcgtgggtgaccatg 480 tgggtgggca cgcttccctg ctgggggtgc agggggtcca cgtggcagcggccacctgga 540 gccctaatgt gcagcggtta agagcaagcc cctggaagtc agagaggcctggcatggagt 600 cttgcttctt gcaaacgagc cgtgtggaga gagagatagt aaatcaacaaagggaaatac 660 atggtctgtc cgaggatgag ctgccggaga gcaatggtga aagtgaagtgggggaggggg 720 cggggctggg aggaaaagcc ttgtgagaag gtgacacgag agcacggccttgaaggggaa 780 gaaggagggc actatggagg tcccggcgaa gcgtggcctg gccgaggaacggcatgtgca 840 gaggtcctgc cgaggagctc aagacaagta ggggacggtg gggctggagtggagagagtg 900 agtgggagga ggagtaggag tcagagagga gctcaggaca gatcctttaggctctaggga 960 cacgataaac acagtgtttt ttgtcttgtc aagtgtgtcc tttttatttttttgaaagag 1020 tctcgctctg tagcccaggc tggagtgcag cggtgcgacc tcggctcagtgcaacctctg 1080 cctcccgggt ccaagcaatt ctcctgcctc agcctcccga gtagctgggattacaggcac 1140 ccgccaccac gcactgctaa tttttgtatt ttagtagaga ccgggttttgccatgttggt 1200 caggctggtc tcgaactcct gacctcaggt gatccgcccg cctcggcctcccagagtggt 1260 gtgagccact atgccctgca gcacttgtca agtctttctc agcgttcccctcctctccac 1320 tgcagctccc agtgccccag tctgggcctc gtcttcactt cctgggatccctgacattgc 1380 ctgctaggct ctccctgtct ctggtctggc tgccttcact gtaacctccacccagcaggt 1440 acctcttcag cacctcccat gaacccagca gaataccaag ccctggggatgcagcaacga 1500 acaggtagac gctgcactcc agcctgggcg acagagcaag actccgcctgaagaaaaaaa 1560 aaaggaccag gccgggcgcg gtggctcacg cctgtaatcc cagcactttgggaggccgag 1620 gtgggtggat catgaggtca ggagttcaag accagcctgg caaaaatggtgaaaccccgt 1680 ctctactgaa aaatacaaaa attagctggg tgcagtggcg ggcgcctgtagtctcagcta 1740 ctcaggaggc tgaggcagga taattgcttg accccaggag gcagaggttgcagtgaaccg 1800 agatcacgcc actgcactcc agcctgggcg acagagcaag actctgcctcaaaaaaaaga 1860 ataaaaataa aaaaaaggac cagatacaga aaacagaagg agacgtactatgaaggaaat 1920 tggagagctt ttgggatact gagtaactca gggtggcctt tcccaggggacatttagctg 1980 agagatagac ggtatgaaga cctgaccgtt cagaaacagg ggaagaggcagcagcccggg 2040 caaaggcctt tggggcagga aagggcttgg atcactggag aagcagaaagatggccagtg 2100 tgaccagagt gtgacaaagt cagagaaaac caggaagatg gagctggagacacaggcggg 2160 gccagatcac gagggtcctc gcagaccaga gcaagggttt ggattttattccaagtatga 2220 agggaagctg ctgaagtgtg ttttccttta caatttgtag ttgaaatataatatgcaaag 2280 tacacaagtc ttaactatat gtaagcttaa tgaatgtttc catgaaccaaataccgctgt 2340 gcaaccatca ccagctcaag agacgaaccc ttctccctcc tcctgactgccagtaacata 2400 gtggttcagc tcaagaaaca gaactcttct gacttcccct aacatagcgggttttctttt 2460 ttgttttgtt ttttgttgtt ttttaagaga caatgtcttt attatttttattttttttta 2520 tttttgagac ggagtcttgc tgtcgcccag gctggagtgc agtggtgcgatctcggctca 2580 ctgcaggctc tgccccccgg ggttcatgcc attctcctgc ctcagcctccctagcagctg 2640 ggactacagg tgcccgccac ctcgcccggc tatttttttg tatttttagtggagacgggg 2700 tttcaccgtg ttagccagga tggtctcgat ctcctgacct cgtgatccgcccacctcggc 2760 ctcccaaagt gctgggatta caggcatgag ccaccgcgcc cagccaagagacacggtctt 2820 gctctgtcgc ccaggctgga tggagtgccg tggtgcgatc acagctcgcggcagccttga 2880 catcctgggc tcaagcaacc ttcctgcctt ggcctcccaa atgttgggattataggcatg 2940 agccactgtg cttggcatct attcatcttt aatgtcaagc aggcaattgaatatttgatc 3000 agggatagaa ttgtctattt gggggtatgc agatgtgctt catgtcatggaactgggccg 3060 ggcgcggtgg ctcatgccta taatcccagc actttgggag gccgaggcaggcggatcata 3120 aggtcaggag atcgagacca tccgggccaa caggtgaaac cccgtctcttactaaaaata 3180 caaaaattag gcaggtgtgg tggtgcgtgc ctgtagtccc agctactcagggaggctgag 3240 acaggagaat tgattgaacc tgggaggcag aggttgtagt gagccaagatcgcgccactg 3300 cactccagcc tgggcgacat gagcgagact ccgtctcaaa aataaacaaaaaaaagtcat 3360 ggaattgatg gaaattgcct aaggggagat gtagaagaaa aggggtctcaggatcaagcc 3420 agcagagaag gcagaaaagg taaggtgtgt gaggtggcag aaaaagggaagagtgtggac 3480 agtgagggtt tcaaggagga ggaactgtct actgcctcct gccaaggacggaggtgtcca 3540 ctgccagttg acataaggtc acccatgaac ttggtgacag gaatttcagtggagaagtgg 3600 ccacagacac aagtctagaa ttgaaatggg agccgaggca gcgtagacaaaagaggaaac 3660 tgctccttgc agagcggctc tgagcgagca ccgagaaatg ggcagtggctttaggggatg 3720 tagcgtcaag gaagtgtctt ttaaagaagt cgggggccgg gcacggtggctcacgcctgt 3780 agtcccagca ctttgggagg ccgaggcagg cagatcactt gaggtcaggagttcgagacc 3840 agcctggcta acacgatgaa accccgtctc tactaaaaat acaaaaaattagctgggcac 3900 ggtggctcgt gcctgtaatc ccagcacttt gggaggcaga ggtgggcagatcacttgagg 3960 tcaggagttt gagaccagcc tagccaacat ggtgaaaccc catctctactaaaactacaa 4020 aaattagccg ggagtggtgg cacgtgcctg taatcccagc cagtcaggaggctgaggcag 4080 gagaatcact ggaatcctgg aggtggaggt ggcagtgagc cgagatggtacctctgtact 4140 ccagcctggg ggacagagtg agactccgtc tcaaaaaaaa aagaaggtggggaaggatct 4200 ttgagggccg gacacgctga ccctgcagga gaggacacat tcttctaacaggggtcggac 4260 aaaagagaac tcttctgtat aatttatgat tttaagattt ttatttattattatttttta 4320 tagaggcaag catttttcac cacgtcaccc aggctggtct ccaactcctgggctcaagtg 4380 tgctgggatt atagccatga gtcaccacac ctggcccaga aactttactaaggacttatt 4440 taaatgattt gcttatttgt gaataggtat tttgttcacg tggttcacaactcaaaagca 4500 acaaaaagca cccagtgaaa agccttcctc tcattctgat ttccagtcactggattctac 4560 tcttgggatg cagtgttttt catctctttt ttgtatcctt ttggaaatagtattctgctt 4620 taaaaagcaa ttacaggcca ggtatggtgg ctcactcctg taatcccagcactttgggag 4680 gccgaggcag gtgatcacct aaggtcagga gttcaagacc agcctggccaatatggtgaa 4740 accctgtctg taccaaaaga caaaaacaaa aacaaaaaca aaaattagccgggcgtggtg 4800 gcgtgctcct gtaatcccag ctactcagga ggctgaggca ggagaatcgcttgaacctgg 4860 gaggcagagg ttgcagtgag ccgagattgt gccactgtac tccagcctgggccacagagc 4920 aaggttccat ctcaaacaaa acaaaacaaa acaaacaaaa aaacaaaacaaaagctaata 4980 caaacacata tacaatagac aaaactgtaa atattttatt atttttattttttttagtag 5040 agacagggtt tcaccatgtt ggccaggatg gtctcaaact cctgacctcaggtgatccac 5100 ccacctcagc ctcccgatag ttaggattac aggcatgagc caccacacccggcctaaaat 5160 tgtaaacgtt ttagaagaaa gtatagatga atcccttcgt gatctcggggaagaagagat 5220 tttttaaaaa agataccaaa agaagcacaa attataaaag aaaagattgaaaatgttggt 5280 gttaaaatta aaaacttgtt ttaaaacaag cttgtgtaac ccatgacccacaggctgcat 5340 gtggcccaga aaagctttga ctgcagccca acacaaattc gtaaactttcctaaaacatt 5400 atgagatttt ttttgagatt ttgttttgtt ttgttttttg tttttttagctcattcggta 5460 tcattaatgt tagcatattt tacgtggggc ccaagacaat tcttcttccaatgtgtctca 5520 ggggagccaa aagattggac acccctgcca taaacatgaa aagacaatggccgggcacgg 5580 tggctcacgc ctgtaatccc agcactttgg gaggctgagg ggggcgggatcacctgaggt 5640 caggagtttg agacaagcgt gaccaatgtg gtgaaaccct gtctctactaaaaatacaaa 5700 aattagccgg gcatgctcgt gcacacctat agtcccaact actcagcagggtgaggcagg 5760 agaacctctt gaacccggga agcggaggtt gcagtgagcc gacattgcacccctgcactc 5820 gagcctgggt gacagagtga gtctccactg gaaaaaaaaa aaaaagaacagtgtgataca 5880 ttgacctaag gtttaagaac atgcaaactg atactatata tcacttagggacaaaaactt 5940 acatggtaaa agtaaaaaga aatgtacgaa aataataaaa atcaaattcaagatggtggt 6000 tatggtgacg ggaaagaact gaggcggaaa tataaggttg tcactatattgagaaatttt 6060 tctatctttt ttttcttttt tcttttttga gacggggtct cgctctgtcgcccaggatgg 6120 agtgcagtgg tgtgatctca gctcactgca acctccgcct cccaggtttaagtgattctc 6180 ctgcctcaga ctcccaagta gctgggacta caggtgcgcg ccaacacacctgggtaattt 6240 tgtttgtatt tttagtagag atggggtttc accgtgttga ctaggctggtctcgaactcc 6300 tgacctcagg tgatcccccg gcctcggtct cccaaagtgc tgggataacaagcgtgagcc 6360 actgcgccca gctttgtttg catttttagg tgagatgggg tttcaccacgttggccaggc 6420 tggtcttgaa ctcctgacct caggtgatgc acctgcctca gtctcccaaagtgctggatt 6480 acaggcgtta gcccctgcgc ccggcccctg aaggaaaatc taaaggaagaggaaggtgtg 6540 caaatgtgtg cgccttaggc gtaatggatg gtggtgcagc agtgggttaaagttaacacg 6600 agacagtgat gcaatcacag aatccaaatt gagtgcaggt cgctttaagaaaggagtagc 6660 tgtaatctga agcctgctta tggacgctgg attagaaggc agcaaaaaaagctctgtgct 6720 ggctggagcc ccctcagtgt gcaggcttag agggactagg ctgggtgtggagctgcagcg 6780 tatccacagg taaagcagct ccctggctgc tctgatgcca gggacggcgggagaggctcc 6840 cctgggctgg ggggacaggg gagaggcagg ggcactccag ggagcagaaaagaggggtgc 6900 aagggagagg aaatgcgaga cagcagcccc tgcaatttgg ggcaaaagggtgagtggatg 6960 agagagggca gagggagctg gggggacaag gccgaaggcc aggacccagtgatccccaaa 7020 tcccactgca ccgacggaag aggctggaaa ggcttttgaa tgaagtgagtgggaaacagc 7080 ggagggcggg tcatggggag gaaaggggag ctaagctgct gggtcgggtctgagcagcac 7140 cccaagactg gagcccgagg caaggaggct cacgggagct gcttccaccaagggcagtca 7200 ggaaggcggc cgccctgcag cccagccctg gcccctgctc cctcggctccctgctacttt 7260 ttcaaaatca gctggtgctg actgttaagg caatttccca gcaccaccaaaccgctggcc 7320 tcggcgccct ggctgagggc tgggatggag gacagctggg tccttctagccagcccccac 7380 ccactctctt tggctacatg agtcaaggct gggcgaccaa tgaggttgtggcctccggca 7440 aacaatgacc actatttagg ccggcaggtg tatagggcgt gggggcccagctgccagtgc 7500 tggagacaag ggctgtccga gatgaaccct ttctgctgcc tgccaagccactgggagggg 7560 taggtctcag caggattccc agaaaccccg cccctgtcca gcctaggccccccacccggt 7620 gttagctaac ccaacgttag cccccaggtt ccgtggggtt ggggggcagggagtcctatt 7680 cttggggctg ctgcttctgg ggtgtgggga agtgcaactc cacggcaccctgggctgact 7740 cattcagctt ctaaagcttc aggaaacatt gtttggggct gggtcaccatgggtgggcca 7800 gagaggaccc ctcaatcccc tccggagagc caggggaggg ggaggtgcccttccccatgc 7860 tatctccgag gccactgcca tgtggcctga aggctgtgcg gttctgggaagagggggagg 7920 tggcggtgga ggctgtttgt ctcctaactg ggcttaatct gaaacacatgtattggcttg 7980 agttgatccg cctcacgtgg aggcaagatc acaaaagctt ctgtgtttcttgatgtgggc 8040 aattgtcaga aaataaggcc tgaccttggc ccagcaggga gggtatctacctctccctga 8100 gccctccccc gcctgctagg acgagagcgg ggcttggata ctgccctttggacaggatgg 8160 catcattgtc tgtggctgca gccagccagc ggtcgcctgc tcagcccatgagcaaccact 8220 gtggacaggg tattgcgtgt gtgctgaggg gcgtccatgc agacccccacgcttgccctc 8280 tcactgccct tgtagggttt tcaatcatct ctcctcttcc cttatccagatggcttgaag 8340 tggaggattc agacttgccg ttaatactct gggtccctgt gtctagctcggggccacctt 8400 tggacccatg tcccttccct gccaggctcc ctcacctcac ctcagcctacccacattgtg 8460 acaatcatct accacctgat ctggggtttg ggcttagatt ctgtaggcaccaagactaaa 8520 gtcgctcctt caagtccatt tgaattgtga ctttagtttc cttaaatactatgccaggat 8580 aatggccagg gatggtggct cacgcctgta ctcctggcac tttgggatgctggtggatca 8640 cctgagatca ggattccagg ccagcctggc caacacggtg aaaccccatctctactaaaa 8700 cataaaaatt aaccaggtgt ggtggcgggc acctgtaatc ccagctactcaggagactga 8760 ggcaggagaa ttgcttgaac ccgggaggtg gaagttgcac tgagctgagatcgcgccact 8820 gcactttagc ctgggcgaca agagtgaaac tctgtctcaa aaacaaaaaaaactatgccg 8880 ggatgagcct gtctcctccc ttaatttctt acttgggcca gaggaactagaactaacaac 8940 ttctcttcta gccttgcctc ctgtgtacct cactgaattt ttggtctctaataaaccagt 9000 ctgcagaggc tcaggggagg caggctcctg gcagctgggt ggggctggccccagccgggt 9060 ggagaccagc tgtaggcctg gatggtggtg aggcctctgt cttgcactgcagaaagcttt 9120 tcctgttgtc tacacgaaag ttttctccct gcatgtcagg gcagccacgtgcaagagcag 9180 ctggctggga acgcagaggt ctgcggctcg aggcggggtt tagaaaggaaaaccaggctg 9240 cttcctgctg cccgtcctgc cttaagctga gtaaactcaa aggcaatcttctttcatgcc 9300 tcacgatatt gtccagtgga ttatctgatt taatttgaag gacgagagccaacaatcaca 9360 caacgtcctc ccaaattttc tgatccactt tgttctggga agtcaaaaagtgcgtgtgct 9420 gtgtgggtgg atgtttgtga tataaatgga taatgaagga tgatgtgttggggggccagg 9480 gcaggggaga caacgctgtt cagattctac attttttttt ccttttttttttttttttga 9540 gatggagtct tgctctgttg cccagcctgg agtgcagtgg cgcgatctcagctcactgca 9600 acctccactt cctggattca agtgattctc ctgccttagc ctcccaagtagctgggatta 9660 caggcatgcg ccaccacacc cggctaattt ttgtattttt agtagagatggggtttctcc 9720 atgttggcca ggatggtctc aaactcctga cctcaggtga tctacccgcctcggcctctc 9780 aaagtgctgg gattacaggt ttgagccact gcgcctggcc tttttttttttttttgagat 9840 ggagttttca ctcttgttgc ccaggctgga gtgcagtggt gcgatcttggctcactgcaa 9900 cctccacctc ccaagttcaa gtgattctcc agccttagcc ctccaagtagctgggactac 9960 aggtgtgtgc caccatgcct ggctatttta ttttatttta ttttatttatttatttttga 10020 gactaagtct tgctctgttg cccaggctgg agtgcagtgg cataatcggctcactgcaac 10080 ctctgcctcc caggttcaag tgattctcct gcctcagcct cctgagtaactgggattaca 10140 ggggcctgcc accacgcctg gctacttttt gtatttttag tatagatggggtttcaccat 10200 gttggccagg ctggtctcga actcctgacc tcaggctatc cgcctgcctcagcctcccaa 10260 aagtgctggg attacaggca tgagccactg tgctcggtag ttgtttattttaatagtagg 10320 ttattttatt tccattttac aagagaaaaa atggtgattt aaagagctactaagacacag 10380 cactgagacc atgtgtgatg gcatgcgcct gcagtcccag ctactcacgaggctgaggca 10440 ggaggatcac atgaggtcag gagttccagg ctgtggagtg ctatggttgtgtagtgaata 10500 gccactacac tccagcctgg gcagcacagc aagatcttgt ctcccaaaaaaaaaaaaaaa 10560 aaaaaatttc aaatgtgaac ccaggatctc tgaccgggct aggccctgcactgctaacca 10620 tgggaggaag agctcttgaa agggaactgt gggagaaggg aatgagctgccttgtgaggc 10680 cacagaagtc caaagacagc ttgagaattt ggaggacagc acgtgccggactggggtgcc 10740 tctatgcttg gtatccggtg attccatgga ggagacctgg gttctgccccattctcctgg 10800 gaggggttgc ccaaagtctt atcaccggag tgggtcagct gcctccaggacaaagcttta 10860 gcatacactt gtgctgggcc atactccacg tggagaagcc ctgctggggctggggcccca 10920 ctgctctgga tctttaaaag ctattggttc aggggccagg tgtaatggctcacacctata 10980 accctagcac tttgggaggc tgaagcaggt ggatagcctg aggtcaggagtttgagacaa 11040 gcctgatgac gtggtgaaac cccatcgcta ttaaaaatac aaaaaattagccgggcatgg 11100 tggcaggtgc ctgtaattcc agctacttgg gaggctgagg cgggagaatcgcttgaaccc 11160 aggaggcgga ggttgcagtg agccaagatc gctccactgt actccagcctgggcgacaga 11220 gccagactct gtttcaaaaa ataaaatata aataaataaa taaataaataaataaataaa 11280 taaaagcttt aggcttaaag gagggtcccc tgacgcagac agtggaacaaaagcacaagc 11340 ttatggtatg actgtgggcc ctgaggcagg gggaggggcg ggagaaccttgctgggaggg 11400 atgggccatc aagctgaggg tccacttctg ggggcctgga ggggtgaggggtggtcgctg 11460 cagggggtgg gggaaagtga ctaacccctg ggtcctggct gggcctggctggggtggcca 11520 ggaaggggta gcggggcagt gcagtgtcgg gggagagcgg cttgctgcctcgttcttttc 11580 ttgcaggccc caggatgcag gccctggtgc tactcctctg cattggagccctcctcgggc 11640 acagcagctg ccagaaccct gccagccccc cggaggtcag taggcaggcggggagggcgt 11700 ggtcagcatt ccccgcccct ccttggcagg cagcacggga aacaggacagggaacccgga 11760 cccaggttcc aggccaggct tgggccttta tttctctagg gctggagtttctccagcagc 11820 aaaacagaga gaaaatgtct tgccttgcct ttcaggggat ggagtagggacatgaataag 11880 atcccaaaag agtaaaaatc tgaagcactt ttaacaagtc cagggcaattctcctgcctc 11940 agcttcccaa gcagctggga ttacaggcat gcaccaccaa gcccggctcattttgtattt 12000 ttagtagaga cggggtttct ccatgttggt caggctggtc tcgaactcccgacctcaagt 12060 gattctcctg cctcggcctc ccaaagtgcc gggatgacag gtgtgagccaccgcacctgg 12120 ccaggatctt ttctcattac cttgtcttcc tagtgggggc tccactgagcaggtcatgtt 12180 cccggacatt tgttcggata ctgaccaggc tgtggcaggg agtgagggtatggagtgacc 12240 tctctcctgc ccagaaaggg cgcagctggg ttcccaaggc agatacaggcacatggaggg 12300 aagcctgggc catatgagtg ttatggggtg agtgttggcg gaggcccacccttgagggac 12360 aagagcagct gggcatcttg gcgagagccc tggactttcg tgaggtcagagtatgaattc 12420 tgcgtctccc tcttcctagc tttgtgaccc tagacaaccc ttacctcagtctttgcttcc 12480 ttgcctatga aatgggataa aaacacccat tctacagggc catgtggccactcatttatt 12540 tctcatctac caaacaccta ctcgacaggg gctggcaatg ggcggaaataaaaactcagt 12600 tctgccgggt gcggtggctc acacctgtaa tcccagcagt gtgggaggcggagcaggacg 12660 atcccttgaa tccaggagtt tgagaccagc ataggcaaca tagtgagacccctgtctcta 12720 cacaaaagca aaaattacca ggcgtggtgg caagtgcttg tggtactacctacttgggaa 12780 gctgaggtgg gaggatcact tgagcccagg agattaagac tgcagtgaggggccgggcgc 12840 ggtggctcac gcctgtaatc ccagcacttt gggaggtgga ggtgggtggatcacgaggtc 12900 aggagatcga gaccatcctg gctaacacgg tgaaaccccg tctctactaaaaatacaaaa 12960 aattagctgg gtgtggtggg gggcgcctgt agtcccagct actcgggaggctgaggcagg 13020 agaatggcgt gaacccggga ggtggaggtt gcagtgagct gagctcgcaccactgcactc 13080 cagcctgggc gacagagtga gactccgtct caaaaaaaaa aaaaaaagaaagaaagaaag 13140 aaaaactgag ttcttttttt taactttctt tttttagaga cagagtctcactccatcacc 13200 catgctggag tacagtggtg cgatcttggc tcactgcaat cttggcctcctgagttcaac 13260 caattctcat gcctcagcct cccaaatagc tgggaccaca ggcacgtgccaccacgccca 13320 gctaattttt tgggtatttt tagtagagat ggggcctcac catgttgctcaggttggtct 13380 gaaactcctg agctcaagtg atccatcttc ctcggcctgc caaagtgctgggattatagg 13440 cataagccac tgcacctagc tcccaatttt tatatttata tttatttttatttacttatt 13500 tattttttga gacagggtct cactctgtca cccaggctgg agtacagtggcactatctca 13560 gctcactgca acctctgcct cctgggttca agcgaatctc gtgcctcagcctcctgagta 13620 gctgggatta caggcatgca ccaccatgcc ccgttaattt ttttgtatttttagtagaga 13680 cgggtttcac cgtgttgccc aggatggtct cgaactcctg acctcaagtgattcacccac 13740 ctcagcctcc caaagtgctg ggattatagg tgtgagccac tcggctgatggtttttaaaa 13800 agtgggtcat ggggctgggc gcggtggctc atgcctgtaa tcccagcactttggtagacc 13860 gaggcgggtg gatcacaagg tcaggagatc gagaccatcc tgcctaacacggtgaaaccc 13920 cgtctctact aaaaatacaa aaaattaccc aggcatggtg gtgggcgcctgtagtcccag 13980 ctactcggga ggctgaggca ggagaatggc gtgaacctgg gaggcggagcttgcagtgag 14040 ccgagatcac gccaccgtac tccagcctga gcgacagagc gagactccgtctcaaaaaaa 14100 aaaaaaaaaa gtgggtcata ggtttcggct tataggtcac aagtgtttaaacctggccat 14160 gaggccaggc gcagtggcgc atgcctgtaa tcccagccat ttgggaggctaaggcaggaa 14220 aatcgcttga accggggagg tggaggttgc agtgagctga gatcgcgccactgaactcta 14280 gcctgggtga cacagtaaga ctctgtctca aataaaaaaa aaaacagctgatctctcttc 14340 tgcgctgtct ctccacagag agctcatgcg tgatcaggga gtaaaactcattcccgtttt 14400 aggccaaaca cagaaaaatt aggaaggaca gccccaaggg gccagaaccaccaccctaca 14460 caaagccgtg aggagacagt ccctgtgcat ctctgcgagt ccctgaactcaaacccaaga 14520 cttcctgtct cctgccaggg ctccccagac cccgacagca caggggcgctggtggaggag 14580 g 14581 <210> SEQ ID NO 5 <211> LENGTH: 5262 <212>TYPE: DNA <213> ORGANISM: HUMAN <220> FEATURE: <223> OTHER INFORMATION:EXON 35-161; EXON 1142-1297; EXON 1984-2187; EXON 5170-5255; INTRON162-1141; INTRON 1298-1983; INTRON 2188-5169; CDS 35-161; CDS 1142-1297;CDS 1984-2187; CDS 5170-5255 <221> NAME/KEY: exon <222> LOCATION:(35)..(160) <221> NAME/KEY: exon <222> LOCATION: (1142)..(1297) <221>NAME/KEY: exon <222> LOCATION: (1984)..(2187) <221> NAME/KEY: exon <222>LOCATION: (5170)..(5256) <221> NAME/KEY: intron <222> LOCATION:(162)..(1141) <221> NAME/KEY: intron <222> LOCATION: (1298)..(1983)<221> NAME/KEY: intron <222> LOCATION: (2188)..(5169) <223> OTHERINFORMATION: n = a or g or t or c, any base <400> SEQUENCE: 5 acaagctggcagcggctgtc tccaacttga atac aag ctg gca gcg gct gtc tcc 55 aac ttc ggctat gac ctg tac cgg gtg cga tcc agc atg agc ccc acg 103 acc aac gtg ctcctg tct cct ctc agt gtg gcc acg gcc ctc tcg gcc 151 ctc tcg ctgggtgagtgct cagatgcagg aagccccagg cagacctgga 200 gaggccccct gtggcctctgcgtaaacgtg gctgagttta ttgacatttc agttcagcga 260 ggggtgaagt agcaccaggggcctggcctg ggggtcccag ctgtgtaagc aggagctcag 320 gggctgcaca cacacgattccccagctccc cgaaaggggc tgggcaccac tgacatggcg 380 cttggcctca gggttcgcttattgacacag tgacttcaag gcacattctt gcattcctta 440 accaagctgg tgctagcctaggttcctggg atgtaactgc aaacaagcag gtgtgggctt 500 gccctcaccg aggacacagctgggttcaca ggggaactaa taccagctca ctacagaata 560 gtcttttttt tttntttttttnnnctttct gagacggagt ctcgctttgt cnccaaggct 620 ggagtgcagt ggtgtgatctcagctcactg caacctctgc ctccctggtt caaggaattc 680 tcctgcctca gcctccagagtagctgggat tacaggcacc tgccatcatg cccagctaat 740 ttttgtattt ttagtagagacggggtttca ccatgttgcc taggctggtc tcaaactccc 800 gggctcaagc gatccacccgccttggcctc ccaaagtgct gggattacag gcgtgagcca 860 ccgcgcctgg ccagaataatcttaagggct atgatgggag aagtacaggg actggtacct 920 ctcactccct cactcccaccttccaggcct gatgccttta acctacttca ggaaaatctc 980 taaggatgaa nattccttggccacctagat tgtcttgaag atcagcctac ttgggctctc 1040 agcagacaaa aaagatgagtatagtgtctg tgttctggga gggggcttga tttggggccc 1100 tggtgtgcag ttatcaacgtccacatcctt gtctctggca g gag cgg agc agc gaa 1156 cag aat cca tca ttc accggg ctc tct act atg act tga tca gca gcc 1204 cag aca tcc atg gta cct ataagg agc tcc ttg aca cgg tca ctg ccc 1252 ccc aga aga acc tca aga gtg cctccc gga tcg tct ttg aga aga 1297 gtgagtcgcc tttgcagccc aagttgcctgaggcatgngg gntccatgct gcaggctggg 1357 ggggtctttt tttttttttt nnnnagacggagtctcgctc tgttgcccag gctggagtgc 1417 agtggcgnga tctcggctca ctgcaacctccacctcccgg gttcacacca tcctcctgcc 1477 tcagcctccc gagtagctgg gactgcaggngcccagctaa tctttnttgt atttttagca 1537 gagacggggt ttcaccgtgt ttgccaggatagtctcgatc tcctgacctg gtgttctgcc 1597 cgcctcgacc tcccaaagtg ctgggattacaggtgtgagc caccgcgctc ggcccgtttc 1657 taaacaatag atcatgtgtg cccaggcctggcctggcact ggtgtggagg aagggcccgt 1717 gagcccaaag aggctcagaa agaggaagtgggctgcagga gacggtggga ggggcnggga 1777 gggcagtggc gcgatgtggg gaaatctgctgcccccctgg ccagtgcctg gggatgccag 1837 cagaagtcct ggcaagtcac aggaagatgctggctgggaa gtcagggcct gctgagcgct 1897 aaaccagaac ccgagcctgg caggctctcaaagacgggat gcttgtcgtn gagtctcata 1957 ngctaacctc tgctccgcct cttctc agctac gca taa aat cca gct ttg tgg 2010 cac ctc tgg aaa agt cat atg gga ccaggc cca gag tcc tga cgg gca 2058 acc ctc gct tgg acc tgc aag aga tca acaact ggg tgc agg cgc aga 2106 tga aag gga agc tcg cca ggt cca caa agg aaattc ccg atg aga tca 2154 gca ttc tcc ttc tcg gtg tgg cgc act tca agggtgagcgcgt ctccaattct 2207 ttttcattta ttttactgta ttttaactaa ttaattaattcgatggagtc ttactctgta 2267 gccctaactg gagtgcagtg gtgcgatctc agctcaatgcaacctccgcc tcccaggttc 2327 aagcaattct tgtgcctcag cctcccgagt agctgggattacagggatgt accaccactc 2387 ccggctaatt ttttgtattt aatagacatg gggtttcaccatgttggcca ggctggtctc 2447 gaactcctga gctcaggtgg tctgcccgcc tcagcctcccaaagtgctag gattacaagc 2507 ttgagccacc acgcccagcc ctttttattt ttaaattaagagacaaggtg ttgccatgat 2567 gcccaggctg gtctcgaact cctgggctca agtaatcctcccaccttggc ctcccaaagt 2627 gctgggatta caggcatgag ccaccgcgcc cggcccttttacatttattt atttattttt 2687 tgagacagag tcttgctctg tcacccaggc tggagtgcagtggcgcgatc tcggctcact 2747 gcaagctctg ccttccaggt tcacaccatt ctcctgcctcgacctcccga gtagctggga 2807 ctacaggcgc ccgccactgc gccctactaa ttttttgtatttttagtaga gacggggttt 2867 caccgtggtc tcgatctcct gacctcgtga tccacccgcctcagcctccc aaagtgctgg 2927 gattacaggc gtgagccact gcgcccggcc cttttacatttatttttaaa ttaagagaca 2987 gggtgtcact atgatgccga ggctggtctc gaactcctgagctgaagtga tcctcccacc 3047 tcggcctccc aaaatgctgg gattaccatg tccaactttccacttcttgt ttgaccaagg 3107 atggatggca gacatcagaa ggggcttgga aagggaggtgtcaaagacct tgcccagcat 3167 ggagtctggg tcacagctgg gggaggatct gggaactgtgcttgcctgaa gcttacctgc 3227 ttgtcatcaa atccaaggca aggcgtgaat gtctatagagtgagagactt gtggagacag 3287 aagagcagag agggaggaag aatgaacact gggtctgtttggggctttcc cagcttttga 3347 gtcagacaag atttatttat ttatttaaga tggagtctcattctgttgcc caggctggag 3407 tgcagtggtg ccatcttggc tcactacagc ctccccacctcccaggttca agtgcttctc 3467 ctgcctcagc ctcccgagta gttgggatta caggcgcccgccaccacacc cagctaattt 3527 ttgtattttc agtagagatg gggtttcgcc atgctggccaggctgttctc gaaaactcct 3587 gacctcagat gatccacccg cctcggcctc ccacagtgctgggattacag gcgtgagcca 3647 ctgcgctggc caaatcagac aaggtttaaa tcccagctctgcctgtacta gctgaggaac 3707 tctgcacaca tttcataacc tttctgggcc tacgttctcacctttaacgt gaggataata 3767 tatctacttc atagacacct ttttatgttg tctccaagttttctaacagc tctagttctg 3827 tacccaagac atggcaggtg gccaacgaca tccttctaggctgtggtgat gtgtttggag 3887 cttgttccac gggtcttgtg tggggccagc cctgttcagataaggccttg tggggtggcc 3947 tggggtaggg ggaggggttg ggcaaactct cccttaaaacgctttgtaac catctgaggc 4007 accagcaaga gcggcccccg agcctggaca aaatccaaacggcttcctac ttcaagcact 4067 gatgtctagt gagtgaagga acagctctgg gtccaggatattataggtca cattaaacta 4127 aaggggcttg gccatcagct ggcttccaga gcgtcagccagttacttcac ctctttggct 4187 ttggcctgtt ttcagctaca agaggactta atccagaggacctcagaggt ccttcccagc 4247 tcagaccttc tttgactgtc tcccagagac actgctgtaggagtgcacac cagtttactt 4307 ttctttcttt tgtttttgag atggagtttc gctctttttgcctaggctgg agtgctgtgg 4367 tgtgatctca gctcactgca acctctggct cccaggttcaagtgattctc ctgtctctgc 4427 ctcccgagta gctgggatta cagacaccca ccactgcacccggctagttt ttgtattttc 4487 agtagagatg gggtttcgcc atgctggcca ggctgttctcgaaaactcct gacctcagat 4547 gatccatccg ccttggcctc ccaaagtgct gagattacagatgtgaggca ccacacccgg 4607 ccatttttgt atttttagta gagacggggt tttgccatgttggccacgct ggtctcaaac 4667 tcctgacctc aagtgatctg cccaccttgg cctcctgaagggctgggact acaggcgtga 4727 gtcaccgtgc ccggccattt ttgtattttt aggacagcgttttttcatgt tggccaggct 4787 ggtctcaaac tcctgacctc aagtgatcca cccaccccggcctcccaata tgctgggatt 4847 ccaggtgtga gttaccatgc ccggctacca ctttacttttcctgcaggct atcacagaac 4907 gtgtacaatc tagactctaa tcaaccaaat caacgtcttgccatcggagt ttgctggtga 4967 agggcacttg gggtcctgga aataactgta ggctccaagccacacacact gagataggcc 5027 tattccctga ggcctcagag cccctgacag ctaagctcccttgagtcggg caattttcaa 5087 caacgtgctc tggggacaca gcatggcgcc actgtctttctggtctcctg gggctcagac 5147 tatgtcatac acttctttcc ag ggc agt ggg taa caaagt ttg act cca gaa 5199 aga ctt ccc tcg agg att tct act tgg atg aag agagga ccg tga ggg 5247 tcc cca tga tgaatc 5262 <210> SEQ ID NO 6 <211>LENGTH: 4421 <212> TYPE: DNA <213> ORGANISM: HUMAN <220> FEATURE: <223>OTHER INFORMATION: CDS 66-322 <400> SEQUENCE: 6 ggatcccttg gttggggtgttggggaaggc agggttttaa cggaaatctc tctccatctc 60 tacagagctg caatccttgtttgattcacc agactttagc aagatcacag gcaaacccat 120 caagctgact caggtggaacaccgggctgg ctttgagtgg aacgaggatg gggcgggaac 180 cacccccagc ccagggctgcagcctgccca cctcaccttc ccgctggact atcaccttaa 240 ccagcctttc atcttcgtactgagggacac agacacaggg gcccttctct tcattggcaa 300 gattctggac cccaggggcccctaatatcc cagtttaata ttccaatacc ctagaagaaa 360 acccgaggga cagcagattccacaggacac gaaggctgcc cctgtaaggt ttcaatgcat 420 acaataaaag agctttatccctaacttctg ttacttcgtt cctcctccta ttttgagcta 480 tgcgaaatat catatgaagagaaacagctc ttgaggaatt tggtggtcct ctacttctag 540 cctggtttta tctaaacactgcaggaagtc accgttcata agaactctta gttacctgtg 600 ttggataagg cacggacagcttctctgctc tgggggtatt tctgtactag gatcagtgat 660 cctcccggga ggccatttcctgcccccata atcagggaag cctgctcgta aacaacacat 720 ggacagatag gagaggccatttgtaactta aggaaacgga cccgatacgt aaagattctg 780 aacatattct ttgtaaggaggtatgcctat tttacaaagt acagccgggt gtggtggctc 840 atggctataa tcccagcactttgggaggcc gaggcgggcg gatcacctga gatcaggagt 900 ttgagaccag cctgaccaacacggagaaac cccgtctgta ctaaaaatac aaaattagca 960 gggtgtggtg gtacatgcctgtaatcccag ctactgggga ggctgaggca ggagaatcac 1020 ttgaacccgg gaggcggaggttgcagtgag ccgagatcac gccattgcac tccaatctag 1080 gcaataagag caaaactccgtctcaaacaa caaaaaacca aagtataact gggctttttg 1140 aagaacatga aacatgcccagtgtctgaag tagaataact accgaactgt ccgtaggact 1200 aaactttttc ttgaaaaagctctaccaaaa aaagtcaccg gccactccct tgtcacagtt 1260 attagacagg aggagaaatgataattctac tgcccttcat tctacaaatg tttgagtgct 1320 aactgtattc cagattctcaaaaagctatt gccaggtatc tctggggcta ctgatttcct 1380 gatcataatg caatggcaaccaacaggcac ttgggcatgg tgagggtggg caagctttca 1440 aaagcagcgt ggatctggcattcttttcca cgaatgcacc tcaactactt ggcaccagtg 1500 gtaacacagc aaccagggttccgacctaga gaatcccgta accttctgac tggaacgggg 1560 tctgggctgt cgctacacatcctggtggaa ggcagctatc atccctacct tctgccttct 1620 gtctcttaaa tctgaaccacaaacagcaac gtccataccc tcagcattgt tagaatcccc 1680 tgcagcctcc agttctcatactgtctgtat tctactcgcc agtttggaga ggtctggtgg 1740 agaaaaggag tctcttttcaggcttgacaa caaatagaac tcagggccgg gcgcggtggc 1800 tcacgcctgt catcccagcactgtgggagg ccgaagcggg cggatcacct gaggtcggga 1860 gctcaagacc agcctggccaacatggagaa atcccatctt tactaaaaat acaaaattag 1920 ccgggcgtac tggcgaatgcctgtaatgcc agcttctcgg gaggctgagg caggagaatc 1980 gcttgaacct gggaggcagaggttgcggtg agccaagact gtgccactgt actccagcct 2040 tggtgacaga gggagactctgtcttaagaa aaaaagaaaa aaaaaaaaaa agggccgggc 2100 tcacgcctgt aatcccagcactttgggagg ccaaatcacc tgaggccggg agtttgatac 2160 caacctgacc aacatagtgaaatcccgtct ctactaaaaa tacaaaatta gccaggcgtg 2220 gtggcgggcg cctgtaatcccagctactcg ggaggctgaa gcaggagaat cacttgaacc 2280 cggaaggcgg aggttgccgtaagccaagat cgcgccattg cgctccagcc tgggcaacaa 2340 gagtgaaact ccatctcaaaaacaaaacaa aacaaaacaa aaccaacaac tcagaaggag 2400 gcatatgtgt tataaagtctttactacaac tttgatttta ttagtggttg gttactgact 2460 ctgccaagag tacagaatgaagggcagaga gtaaggactg gaaaactggc aggaaacaca 2520 ctgacagccg tcatccctggaggaaactgc tcaataaaac ggctccatat ttacttctct 2580 ggtcacagtt catactccacgattttaaca aaggagtcga ggaagctaga tactgtaagt 2640 ggaacggtgt gtctctggaggtaagcaggc ttgctgattt cttgttttat aattcttttt 2700 taattacaat gtaactactaagagcttcag ttcccactgg agtggtgcac acatctcatt 2760 actactaaaa ccacaggaatgttccaggga aacagactat catcactgag cgaggtggaa 2820 tccagccaaa accccaggctaacatccaga tgcctgcata tcagctaaaa tccttttaaa 2880 ggacttggaa tctccagatactagttttaa gtcttttctg ggaactggga gtttgtactg 2940 gaggccactt aactatttcaaaaaatattc accaaaatag gtgtctctct gactgcaacg 3000 gtttgagtcc tcctcagccctcatatccta ggcttcggac tgttgggaaa gtcttatctt 3060 cctgacgaaa gctcagcagcaacagaacct gttatttttt tgttgagaca gggtcttact 3120 ctgtcaccca ggctggagtgcagtagtgcg atcttggctc actgcagcct cagcctacca 3180 ggctcaggtg accctatctcagcttctcga gtaggtggga ctacaggcat gtgccaccat 3240 gctcggtgaa ctaaacaaacttttttgtag tgatacggtc tcactatatt gcccaggctg 3300 gttttgaact cctgggctcaagtgatcctc ccacctcagc gtctcaaagt actgggatta 3360 caggtgtgag cctctacactgggcctgcag aacctacaca gaatccgcac ctggtctgca 3420 gaacccacac ccgacccacagaacccacac ccgacccaca gaacccacat ctggcagcag 3480 aacctcttag tattttttttttttctttga gatggagtct ggctctgtca cccaggctgg 3540 agtgcagtgg cgcgatctcggctcactgca agctcttcct cccgggttca ccccattctc 3600 ctgcctcaac ctcccgagtagctgtgaata caggcgtccg ccaccacgcc cgactaattt 3660 ttttgtattt ttagtagagacggggtttca ccgtgttagc caggatggtc tggatctcct 3720 gacctcgtga tctgcctgcctcggcctccc aaagtgctgg gattacaggc ttgagccacc 3780 gcacccggcc tcttatttttttttttgaga tggagtctca cactgtcacc tgggctggag 3840 tgcagtggag cgatctcggctcactgcaac ctccgcctcc tgggttcaag agattctcct 3900 gcctcagcct cccaagtagctgggattaca ggtgcccacc accacgcctg gctagttttt 3960 tgtattttta gtaaagatggggtttcacca tgttggccag gctggtcttg aactcctgac 4020 atcaggtgat ccgcccaccttagcctccca aagtgctggg attacaggcg tgagccacca 4080 tacctggcca gcaaaacctctttaacttgt gttccatggg ctccttttct gtgggtcaaa 4140 atcctcctgg aaccctacaatgcaggccct acaggggtgg gtggtaagtc caacaaacag 4200 gatttcatct tctggagctcctggatttca tcgtcccatg ggccacagtg cagcgacaga 4260 acctcctcag ctttctgtattgtgctcagg gcttcgggta ctgcaaacct gagccaaggg 4320 aggtaagagg agttagttcactgattcgtg aggcaaatgt taattgaggg cctactcaca 4380 caccgtgaag aatgtaagatcatttctgtc atcaaggatc c 4421 <210> SEQ ID NO 7 <211> LENGTH: 1153 <212>TYPE: DNA <213> ORGANISM: MOUSE <220> FEATURE: <223> OTHER INFORMATION:mRNA: 1-1153 <223> OTHER INFORMATION: CDS 27..1153 “product = ”pigmentepithelial - derived factor“ gene = ”PEDF“ codon_start = 1” <223> OTHERINFORMATION: mat_peptide 27-1153 “product = ”pigment epithelial-derivedfactor“ gene = ”PEDF“ codon_start = 1 <400> SEQUENCE: 7 ggagctgccgcaaccacagt tccgggatgc aggccctggt gctactcctc tggactggag 60 ccctcctggggcacggcagc agccagaacg tccccagcag ctctgagggc tccccagtcc 120 cggacagcacgggcgagccc gtggaggagg aggacccctt cttcaaggtc cctgtgaaca 180 agctggcagcagctgtctcc aacttcggct acgatctgta ccgcctgaga tccggtgcca 240 gcccaacgggcaacgtcctg ctgtctccac tcagcgtggc cacggccctc tcggccctct 300 ctcttggagctgaacatcga acagagtctg tcattcaccg ggctctctac tacgacctga 360 tcaccaaccctgacatccat agcacctaca aggcgctcct tgcctctgtt actgcccctg 420 agaagaacctcaagagtgct tccagaattg tgtttgagag gaaacttcga gtcaaatcca 480 gctttgttgcccctctggag aagtcctatg ggaccaggcc ccggatcctc acgggcaacc 540 ctcgagtagaccttcaggag attaacaact gggtgcaggc ccagatgaaa gggaagattg 600 cccggtccacgagggaaatg cccagtgccc tcagcatcct tctccttggc gtggcttact 660 tcaaggggcagtgggtaacc aagtttgact cgagaaagac caccctccag gattttcatt 720 tggacgaggacaggaccgtg agagtcccca tgatgtcaga tcctaaggcc atcttacgat 780 acggcttggactctgatctc aactgcaaga ttgcccagct gcccttgaca ggaagtatga 840 gcatcatcttcttcctgccc ctgaccgtga cccagaactt gaccatgata gaagagagcc 900 tcacctctgagttcattcat gacatcgacc gagaactgaa gactatccaa gctgtgctga 960 ctgtccccaagctgaagctg agcttcgaag gcgaacttac caagtctctg caggacatga 1020 agctacagtcgttgtttgaa tcacccgact tcagcaagat tactggcaaa cccgtgaagc 1080 tcacccaagtggaacacagg gctgctttcg agtggaatga agagggggca ggaagcagcc 1140 ccagcccaggcct 1153 <210> SEQ ID NO 8 <211> LENGTH: 1490 <212> TYPE: DNA <213>ORGANISM: HUMAN <220> FEATURE: <223> OTHER INFORMATION: mRNA: 1-1490<223> OTHER INFORMATION: CDS: 117-1373; ”PRODUCT =“PIGMENTEPITHELIAL-DERIVED FACTOR” GENE = “PEDF” CODON_START = 1“ <223> OTHERINFORMATION: sig_peptide: 117-170; ”GENE = “pedf” CODON_START = 1“ <223>OTHER INFORMATION: mat_peptide: 171-1370; ”PRODUCT = “PIGMENTEPITHELIAL-DERIVED FACTOR” GENE = “PEDF” CODON_START = 1“ <400>SEQUENCE: 8 ggacgctgga ttagaaggca gcaaaaaaag atctgtgctg gctggagccccctcagtgtg 60 caggcttaga gggactaggc tgggtgtgga gctgcagcgt atccacaggccccaggatgc 120 aggccctggt gctactcctc tgcattggag ccctcctcgg gcacagcagctgccagaacc 180 ctgccagccc cccggaggag ggctccccag accccgacag cacaggggcgctggtggagg 240 aggaggatcc tttcttcaaa gtccccgtga acaagctggc agcggctgtctccaacttcg 300 gctatgacct gtaccgggtg cgatccagca cgagccccac gaccaacgtgctcctgtctc 360 ctctcagtgt ggccacggcg ctctcggccc tctcgctggg agcggagcagcgaacagaat 420 ccatcattca ccgggctctc tactatgact tgatcagcag cccagacatccatggtacct 480 ataaggagct ccttgacacg gtcactgccc cccagaagaa cctcaagagtgcctcccgga 540 tcgtctttga gaagaagcta cgcataaaat ccagctttgt ggcacctctggaaaagtcat 600 atgggaccag gcccagagtc ctgacgggca accctcgctt ggacctgcaagagatcaaca 660 actgggtgca ggcgcagatg aaagggaagc tcgccaggtc cacaaaggaaattcccgatg 720 agatcagcat tctccttctc ggtgtggcgc acttcaaggg gcagtgggtaacaaagtttg 780 actccagaaa gacttccctc gaggatttct acttggatga agagaggaccgtgagggtcc 840 ccatgatgtc ggaccctaag gctgttttac gctatggctt ggattcagatctcagctgca 900 agattgccca gctgcccttg accggaagca tgagtatcat cttcttcctgcccctgaaag 960 tgacccagaa tttgaccttg atagaggaga gcctcacctc cgagttcattcatgacatag 1020 accgagaact gaagaccgtg caggcggtcc tcactgtccc caagctgaagctgagttacg 1080 aaggcgaagt caccaagtcc ctgcaggaga tgaagctgca atccttgtttgattcaccag 1140 actttagcaa gatcacaggc aaacccatca agctgactca ggtggaacaccgggctggct 1200 ttgagtggaa cgaggatggg gcgggaacca cccccagccc agggctgcagcctgcccacc 1260 tcaccttccc gctggactat caccttaacc agcctttcat cttcgtactgagggacacag 1320 acacaggggc ccttctcttc attggcaaga ttctggaccc caggggcccctaatatccca 1380 gtttaatatt ccaataccct agaagaaaac ccgagggaca gcagattccacaggacacga 1440 aggctgcccc tgtaaggttt caatgcatac aataaaagag ctttatccct1490 <210> SEQ ID NO 9 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: SYNTHETIC PRIMER <223> OTHER INFORMATION: PRIMER603 <400> SEQUENCE: 9 acaagctggc agcggctgtc 20 <210> SEQ ID NO 10 <211>LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:SYNTHETIC PRIMER <223> OTHER INFORMATION: ANTISENSE PRIMER 604 <400>SEQUENCE: 10 cagaggtgcc acaaagctgg 20 <210> SEQ ID NO 11 <211> LENGTH:20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: SYNTHETICPRIMER <223> OTHER INFORMATION: PRIMER 605 <400> SEQUENCE: 11 ccagctttgtggcacctctg 20 <210> SEQ ID NO 12 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: SYNTHETIC PRIMER <223> OTHERINFORMATION: ANTISENSE PRIMER 606 <400> SEQUENCE: 12 catcatggggaccctcacgg 20 <210> SEQ ID NO 13 <211> LENGTH: 20 <212> TYPE: DNA <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: SYNTHETIC PRIMER <223> OTHERINFORMATION: PRIMER 2213 <400> SEQUENCE: 13 aggatgcagg ccctggtgct 20<210> SEQ ID NO 14 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: SYNTHETIC PRIMER <223> OTHER INFORMATION:ANTISENSE PRIMER 2744 <400> SEQUENCE: 14 cctcctccac cagcgcccct 20 <210>SEQ ID NO 15 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: SYNTHETIC PRIMER <223> OTHER INFORMATION: PRIMER2238 <400> SEQUENCE: 15 atgtcggacc ctaaggctgt t 21 <210> SEQ ID NO 16<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: SYNTHETIC PRIMER <223> OTHER INFORMATION: ANTISENSE PRIMER 354<400> SEQUENCE: 16 tggggacagt gaggaccgcc 20 <210> SEQ ID NO 17 <211>LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:SYNTHETIC PRIMER <223> OTHER INFORMATION: PRIMER JT10-UP01 <400>SEQUENCE: 17 ggtgtgcaaa tgtgtgcgcc ttag 24 <210> SEQ ID NO 18 <211>LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:SYNTHETIC PRIMER <223> OTHER INFORMATION: PRIMER JT10-DP01 <400>SEQUENCE: 18 gggagctgct ttacctgtgg atac 24 <210> SEQ ID NO 19 <211>LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:SYNTHETIC PRIMER <223> OTHER INFORMATION: PRIMER 1590 <400> SEQUENCE: 19ggacgctgga ttagaaggca gcaaa 25 <210> SEQ ID NO 20 <211> LENGTH: 19 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: SYNTHETIC PRIMER <223>OTHER INFORMATION: PRIMER 1591 <400> SEQUENCE: 20 ccacacccag cctagtccc19 <210> SEQ ID NO 21 <211> LENGTH: 19 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: SYNTHETIC PEPTIDE <400> SEQUENCE: 21 Thr Ser LeuGlu Asp Phe Tyr Leu Asp Glu Glu Arg Thr Val Arg Val 1 5 10 15 Pro MetMet <210> SEQ ID NO 22 <211> LENGTH: 26 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: SYNTHETIC PEPTIDE <400> SEQUENCE: 22 Ser Tyr GlyThr Arg Pro Arg Val Leu Thr Gly Asn Pro Arg Leu Asp 1 5 10 15 Leu GlnGlu Ile Asn Asn Trp Val Gln Ala 20 25 <210> SEQ ID NO 23 <211> LENGTH:14 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE:<223> OTHER INFORMATION: Description of Artificial Sequence: SYNTHETICPEPTIDE <400> SEQUENCE: 23 Leu Ala Ala Ala Val Ser Asn Phe Gly Tyr AspLeu Tyr Arg 1 5 10 <210> SEQ ID NO 24 <211> LENGTH: 29 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: SYNTHETIC PEPTIDE <400>SEQUENCE: 24 Ala Leu Tyr Tyr Asp Leu Ile Ser Ser Pro Asp Ile His Gly ThrTyr 1 5 10 15 Lys Glu Leu Leu Asp Thr Val Thr Ala Pro Gln Lys Asn 20 25<210> SEQ ID NO 25 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: SYNTHETIC PEPTIDE <400> SEQUENCE: 25 Thr Val GlnAla Val Leu Thr Val Pro Lys 1 5 10 <210> SEQ ID NO 26 <211> LENGTH: 11<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: SYNTHETIC PEPTIDE<400> SEQUENCE: 26 Leu Lys Leu Ser Tyr Glu Gly Glu Val Thr Lys 1 5 10<210> SEQ ID NO 27 <211> LENGTH: 30 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: SYNTHETIC PEPTIDE <400> SEQUENCE: 27 Ala Gly PheGlu Trp Asn Glu Asp Gly Ala Gly Thr Thr Pro Ser Pro 1 5 10 15 Gly LeuGln Pro Ala His Leu Thr Phe Pro Leu Asp Tyr His 20 25 30 <210> SEQ ID NO28 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: SYNTHETIC OLIGONUCLEOTIDE <223> OTHER INFORMATION: Y means TOR C; S means G or C <400> SEQUENCE: 28 agyaayttyt aygayctsta 20 <210>SEQ ID NO 29 <211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: SYNTHETIC OLIGONUCLEOTIDE <223> OTHER INFORMATION:Y means T OR C; S means G or C; R means G or A <400> SEQUENCE: 29ctytcytcrt csagrtaraa 20 <210> SEQ ID NO 30 <211> LENGTH: 20 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: SYNTHETIC PRIMER <223>OTHER INFORMATION: PRIMER 353 <400> SEQUENCE: 30 ctgggagcgg acgagcgaac20 <210> SEQ ID NO 31 <211> LENGTH: 21 <212> TYPE: DNA <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: SYNTHETIC PRIMER <223> OTHER INFORMATION: PRIMER1 <400> SEQUENCE: 31 caccttaacc agcctttatt c 21 <210> SEQ ID NO 32 <211>LENGTH: 22 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:SYNTHETIC PRIMER <223> OTHER INFORMATION: PRIMER 2 <400> SEQUENCE: 32aaccttacag gggcagcctt cg 22 <210> SEQ ID NO 33 <211> LENGTH: 24 <212>TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: SYNTHETIC PRIMER <400>SEQUENCE: 33 tatcccagtt taatattcca atac 24 <210> SEQ ID NO 34 <211>LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:SYNTHETIC PRIMER <223> OTHER INFORMATION: ANTISENSE PRIMER 499 <400>SEQUENCE: 34 ttgtatgcat tgaaacctta cagg 24

What is claimed is:
 1. A method of treating retinal disease in a subject comprising administering an effective amount of Pigment Epithelium Derived Factor (PEDF) to said subject, wherein said PEDF comprises SEQ ID NO: 2 or SEQ ID NO:
 3. 2. The method of claim 1, wherein said retinal disease is selected from the group consisting of retinal tumors, neuronal-retinal tumors, macular degeneration, retinitis pigmentosa, retinal detachment, diabetic retinopathy and inherited and age-related pathologies.
 3. The method of claim 2, wherein said tumor is a retinoblastoma.
 4. The method of claim 1, wherein said administration is selected from the group consisting of intravitreal, subretinal or intramuscular administration.
 5. The method of claim 1, wherein said PEDF is in a pharmaceutically acceptable vehicle.
 6. The method of claim 5, wherein said pharmaceutically acceptable vehicle is a saline solution.
 7. The method of claim 1, wherein said effective amount is from about 0.01 to about 10 μg of PEDF at a concentration of about 1 to 100 μg/ml in a saline solution.
 8. The method of claim 1, which further comprises administering said PEDF in conjunction with a different active agent.
 9. The method of claim 1, wherein said PEDF is administered in a time-released compound.
 10. The method of claim 9, wherein said time-released compound is polylactic acid. 